The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts.

Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600­13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration: ClinicalTrials.gov NCT01087307.

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage.

The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H(2)O(2)-induced DNA damage. UVC and H(2)O(2) treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G(0), G(1) and S-phase. Rad18 was important for repressing H(2)O(2)-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G(1), indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H(2)O(2)-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H(2)O(2)-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G(1). In contrast with G(1)-synchronized cultures, S-phase cells were H(2)O(2)-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G(1) (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.

Control of skin cancer by the circadian rhythm.

Skin cancer is the most common form of cancer in the United States. The main cause of this cancer is DNA damage induced by the UV component of sunlight. In humans and mice, UV damage is removed by the nucleotide excision repair system. Here, we report that a rate-limiting subunit of excision repair, the xeroderma pigmentosum group A (XPA) protein, and the excision repair rate exhibit daily rhythmicity in mouse skin, with a minimum in the morning and a maximum in the afternoon/evening. In parallel with the rhythmicity of repair rate, we find that mice exposed to UV radiation (UVR) at 4:00 AM display a decreased latency and about a fivefold increased multiplicity of skin cancer (invasive squamous cell carcinoma) than mice exposed to UVR at 4:00 PM. We conclude that time of day of exposure to UVR is a contributing factor to its carcinogenicity in mice, and possibly in humans.

Gene expression signatures but not cell cycle checkpoint functions distinguish AT carriers from normal individuals.

Ataxia telangiectasia (AT) is a rare autosomal recessive disease caused by mutations in the ataxia telangiectasia-mutated gene (ATM). AT carriers with one mutant ATM allele are usually not severely affected although they carry an increased risk of developing cancer. There has not been an easy and reliable diagnostic method to identify AT carriers. Cell cycle checkpoint functions upon ionizing radiation (IR)-induced DNA damage and gene expression signatures were analyzed in the current study to test for differential responses in human lymphoblastoid cell lines with different ATM genotypes. While both dose- and time-dependent G1 and G2 checkpoint functions were highly attenuated in ATM-/- cell lines, these functions were preserved in ATM+/- cell lines equivalent to ATM+/+ cell lines. However, gene expression signatures at both baseline (consisting of 203 probes) and post-IR treatment (consisting of 126 probes) were able to distinguish ATM+/- cell lines from ATM+/+ and ATM-/- cell lines. Gene ontology (GO) and pathway analysis of the genes in the baseline signature indicate that ATM function-related categories, DNA metabolism, cell cycle, cell death control, and the p53 signaling pathway, were overrepresented. The same analyses of the genes in the IR-responsive signature revealed that biological categories including response to DNA damage stimulus, p53 signaling, and cell cycle pathways were overrepresented, which again confirmed involvement of ATM functions. The results indicate that AT carriers who have unaffected G1 and G2 checkpoint functions can be distinguished from normal individuals and AT patients by expression signatures of genes related to ATM functions.

A predictive mathematical model of the DNA damage G2 checkpoint.

A predictive mathematical model of the transition from the G2 phase in the cell cycle to mitosis (M) was constructed from the known interactions of the proteins that are thought to play significant roles in the G2 to M transition as well as the DNA damage- induced G2 checkpoint. The model simulates the accumulation of active cyclin B1/Cdk1 (MPF) complexes in the nucleus to activate mitosis, the inhibition of this process by DNA damage, and transport of component proteins between cytoplasm and nucleus. Interactions in the model are based on activities of individual phospho-epitopes and binding sites of proteins involved in G2/M. Because tracking phosphoforms leads to combinatorial explosion, we employ a rule-based approach using the BioNetGen software. The model was used to determine the effects of depletion or over-expression of selected proteins involved in the regulation of the G2 to M transition in the presence and absence of DNA damage. Depletion of Plk1 delayed mitotic entry and recovery from the DNA damage-induced G2 arrest and over-expression of MPF attenuated the DNA damage-induced G2 delay. The model recapitulates the G2 delay observed in the biological response to varying levels of a DNA damage signal. The model produced the novel prediction that depletion of pkMyt1 results in an abnormal biological state in which G2 cells with DNA damage accumulate inactive nuclear MPF. Such a detailed model may prove useful for predicting DNA damage G2 checkpoint function in cancer and, therefore, sensitivity to cancer therapy.

Tipin-replication protein A interaction mediates Chk1 phosphorylation by ATR in response to genotoxic stress.

Mammalian Timeless is a multifunctional protein that performs essential roles in the circadian clock, chromosome cohesion, DNA replication fork protection, and DNA replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller protein called Tipin (Timeless-interacting protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and replication stress through interaction of Tipin with the 34-kDa subunit of replication protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1.

The human intra-S checkpoint response to UVC-induced DNA damage.

The intra-S checkpoint response to 254 nm light (UVC)-induced DNA damage appears to have dual functions to slow the rate of DNA synthesis and stabilize replication forks that become stalled at sites of UVC-induced photoproducts in DNA. These functions should provide more time for repair of damaged DNA before its replication and thereby reduce the frequencies of mutations and chromosomal aberrations in surviving cells. This review tries to summarize the history of discovery of the checkpoint, the current state of understanding of the biological features of intra-S checkpoint signaling and its mechanisms of action with a focus primarily on intra-S checkpoint responses in human cells. The differences in the intra-S checkpoint responses to UVC and ionizing radiation-induced DNA damage are emphasized. Evidence that [6-4]pyrimidine-pyrimidone photoproducts in DNA trigger the response is discussed and the relationships between cellular responses to UVC and the molecular dose of UVC-induced DNA damage are briefly summarized. The role of the intra-S checkpoint response in protecting against solar radiation carcinogenesis remains to be determined.

A novel method for large tree visualization.

SUMMARY: Many genomic and proteomic analyses generate as a result a tree of genes or proteins. These trees are often large (containing tens of thousands of nodes and edges), and need a visualization tool to fully display all the information contained in the tree. Clustering analysis can be performed on these trees to obtain clusters of proteins, and we need an efficient way to visualize the clustering results. We present a novel tree visualization tool to help with such analyses. AVAILABILITY: http://www2.renci.org/~jeff/software/bin/win32/ProteinVis-2.1.6-win32.zip.

The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement.

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m(2) UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

Toxicogenomics: transcription profiling for toxicology assessment.

Toxicogenomics, the application of transcription profiling to toxicology, has been widely used for elucidating the molecular and cellular actions of chemicals and other environmental stressors on biological systems, predicting toxicity before any functional damages, and classification of known or new toxicants based on signatures of gene expression. The success of a toxicogenomics study depends upon close collaboration among experts in different fields, including a toxicologist or biologist, a bioinformatician, statistician, physician and, sometimes, mathematician. This review is focused on toxicogenomics studies, including transcription profiling technology, experimental design, significant gene extraction, toxicological results interpretation, potential pathway identification, database input and the applications of toxicogenomics in various fields of toxicological study.

Lack of extracellular signal-regulated kinase mitogen-activated protein kinase signaling shows a new type of melanoma.

The majority of human melanomas harbor activating mutations of either N-RAS or its downstream effector B-RAF, which cause activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase and the ERK MAPK cascade. The melanoma-relevant effectors of ERK activation, however, are largely unknown. In this work, we show that increased ERK activation correlates strongly with mutational status of N-RAS or B-RAF in 21 melanoma cell lines. Melanoma lines that were wild-type for RAS/RAF showed low levels of ERK activation comparable with primary human melanocytes. Through supervised analysis of RNA expression profiles, we identified 82 genes, including TWIST1, HIF1alpha, and IL-8, which correlated with ERK activation across the panel of cell lines and which decreased with pharmacologic inhibition of ERK activity, suggesting that they are ERK transcriptional targets in melanoma. Additionally, lines lacking mutations of N-RAS and B-RAF were molecularly distinct and characterized by p53 inactivation, reduced ERK activity, and increased expression of epithelial markers. Analysis of primary human melanomas by tissue microarray confirmed a high correlation among expression of these epithelial markers in a heterogeneous sample of 570 primary human tumors, suggesting that a significant frequency of primary melanomas is of this "epithelial-like" subtype. These results show a molecularly distinct melanoma subtype that does not require ERK activation or epithelial-mesenchymal transformation for progression.

Ataxia telangiectasia-mutated dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts.

The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6, and 24 h after treatment with 1.5-Gy IR or sham treatment and were compared with those previously recognized in normal human fibroblasts. Under basal conditions, 160 genes or expressed sequence tags were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. On DNA damage, 1,091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1,811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth, and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison with normal cells. The reduced change in DNA damage response genes and the attenuated repression of cell cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells.

Coupling of human circadian and cell cycles by the timeless protein.

The Timeless protein is essential for circadian rhythm in Drosophila. The Timeless orthologue in mice is essential for viability and appears to be required for the maintenance of a robust circadian rhythm as well. We have found that the human Timeless protein interacts with both the circadian clock protein cryptochrome 2 and with the cell cycle checkpoint proteins Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Down-regulation of Timeless in human cells seriously compromises replication and intra-S checkpoints, indicating an intimate connection between the circadian cycle and the DNA damage checkpoints that is in part mediated by the Timeless protein.

An assessment of prognostic immunity markers in breast cancer.

Tumor-infiltrating lymphocytes (TIL) and immunity gene signatures have been reported to be significantly prognostic in breast cancer but have not yet been applied for calculation of risk of recurrence in clinical assays. A compact set of 17 immunity genes was derived herein from an Affymetrix-derived gene expression dataset including 1951 patients (AFFY1951). The 17 immunity genes demonstrated significant prognostic stratification of estrogen receptor (ER)-negative breast cancer patients with high proliferation gene expression. Further analysis of blood and breast cancer single-cell RNA-seq datasets revealed that the 17 immunity genes were derived from TIL that were inactive in the blood and became active in tumor tissue. Expression of the 17 immunity genes was significantly (p < 2.2E-16, n = 91) correlated with TILs percentage on H&E in triple negative breast cancer. To demonstrate the impact of tumor immunity genes on prognosis, we built a Cox model to incorporate breast cancer subtypes, proliferation score and immunity score (72 gene panel) with significant prediction of outcomes (p < 0.0001, n = 1951). The 72 gene panel and its risk evaluation model were validated in two other published gene expression datasets including Illumina beads array data METABRIC (p < 0.0001, n = 1997) and whole transcriptomic mRNA-seq data TCGA (p = 0.00019, n = 996) and in our own targeted RNA-seq data TARGETSEQ (p < 0.0001, n = 303). Further examination of the 72 gene panel in single cell RNA-seq of tumors demonstrated tumor heterogeneity with more than two subtypes observed in each tumor. In conclusion, immunity gene expression was an important parameter for prognosis and should be incorporated into current multi-gene assays to improve assessment of risk of distant metastasis in breast cancer.

Extracting gene expression patterns and identifying co-expressed genes from microarray data reveals biologically responsive processes.

BACKGROUND: A common observation in the analysis of gene expression data is that many genes display similarity in their expression patterns and therefore appear to be co-regulated. However, the variation associated with microarray data and the complexity of the experimental designs make the acquisition of co-expressed genes a challenge. We developed a novel method for Extracting microarray gene expression Patterns and Identifying co-expressed Genes, designated as EPIG. The approach utilizes the underlying structure of gene expression data to extract patterns and identify co-expressed genes that are responsive to experimental conditions. RESULTS: Through evaluation of the correlations among profiles, the magnitude of variation in gene expression profiles, and profile signal-to-noise ratio's, EPIG extracts a set of patterns representing co-expressed genes. The method is shown to work well with a simulated data set and microarray data obtained from time-series studies of dauer recovery and L1 starvation in C. elegans and after ultraviolet (UV) or ionizing radiation (IR)-induced DNA damage in diploid human fibroblasts. With the simulated data set, EPIG extracted the appropriate number of patterns which were more stable and homogeneous than the set of patterns that were determined using the CLICK or CAST clustering algorithms. However, CLICK performed better than EPIG and CAST with respect to the average correlation between clusters/patterns of the simulated data. With real biological data, EPIG extracted more dauer-specific patterns than CLICK. Furthermore, analysis of the IR/UV data revealed 18 unique patterns and 2661 genes out of approximately 17,000 that were identified as significantly expressed and categorized to the patterns by EPIG. The time-dependent patterns displayed similar and dissimilar responses between IR and UV treatments. Gene Ontology analysis applied to each pattern-related subset of co-expressed genes revealed underlying biological processes affected by IR- and/or UV- induced DNA damage. CONCLUSION: EPIG competed with CLICK and performed better than CAST in extracting patterns from simulated data. EPIG extracted more biological informative patterns and co-expressed genes from both C. elegans and IR/UV-treated human fibroblasts. Using Gene Ontology analysis of the genes in the patterns extracted by EPIG, several key biological categories related to p53-dependent cell cycle control were revealed from the IR/UV data. Among them were mitotic cell cycle, DNA replication, DNA repair, cell cycle checkpoint, and G0-like status transition. EPIG can be applied to data sets from a variety of experimental designs.

ATM requirement in gene expression responses to ionizing radiation in human lymphoblasts and fibroblasts.

The heritable disorder ataxia telangiectasia (AT) is caused by mutations in the AT-mutated (ATM) gene with manifestations that include predisposition to lymphoproliferative cancers and hypersensitivity to ionizing radiation (IR). We investigated gene expression changes in response to IR in human lymphoblasts and fibroblasts from seven normal and seven AT-affected individuals. Both cell types displayed ATM-dependent gene expression changes after IR, with some responses shared and some responses varying with cell type and dose. Interestingly, after 5 Gy IR, lymphoblasts displayed ATM-independent responses not seen in the fibroblasts at this dose, which likely reflect signaling through ATM-related kinases, e.g., ATR, in the absence of ATM function.

Dangerous entanglements.

The promise of stem-cell therapy is long-term repair of damaged organs by transplantation. Inoculum size might be a determinant of successful repair, and efforts are being made to improve conditions for ex vivo expansion of stem cells prior to transplantation. A recent article by Damelin and colleagues raised concerns about this approach, demonstrating that stem cells lack the decatenation G2 checkpoint that preserves genetic stability. Cells that fail to disentangle chromatids prior to mitosis are prone to the types of chromosomal aberrations that are observed in cancer cells. Ex vivo expansion of stem cells might have the unintended consequence of encouraging malignant progression.

Multicenter study of acetaminophen hepatotoxicity reveals the importance of biological endpoints in genomic analyses.

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.

An ATR- and Chk1-dependent S checkpoint inhibits replicon initiation following UVC-induced DNA damage.

Inhibition of replicon initiation is a stereotypic DNA damage response mediated through S checkpoint mechanisms not yet fully understood. Studies were undertaken to elucidate the function of checkpoint proteins in the inhibition of replicon initiation following irradiation with 254 nm UV light (UVC) of diploid human fibroblasts immortalized by the ectopic expression of telomerase. Velocity sedimentation analysis of nascent DNA molecules revealed a 50% inhibition of replicon initiation when normal human fibroblasts were treated with a low dose of UVC (1 J/m(2)). Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and AT-like disorder fibroblasts, which lack an S checkpoint response when exposed to ionizing radiation, responded normally when exposed to UVC and inhibited replicon initiation. Pretreatment of normal and AT fibroblasts with caffeine or UCN-01, inhibitors of ATR (AT mutated and Rad3 related) and Chk1, respectively, abolished the S checkpoint response to UVC. Moreover, overexpression of kinase-inactive ATR in U2OS cells severely attenuated UVC-induced Chk1 phosphorylation and reversed the UVC-induced inhibition of replicon initiation, as did overexpression of kinase-inactive Chk1. Taken together, these data suggest that the UVC-induced S checkpoint response of inhibition of replicon initiation is mediated by ATR signaling through Chk-1 and is independent of ATM, Nbs1, and Mre11.

Patterns of cell cycle checkpoint deregulation associated with intrinsic molecular subtypes of human breast cancer cells.

Genomic instability is a hallmark of breast cancer, contributes to tumor heterogeneity, and influences chemotherapy resistance. Although Gap 2 and mitotic checkpoints are thought to prevent genomic instability, the role of these checkpoints in breast cancer is poorly understood. Here, we assess the Gap 2 and mitotic checkpoint functions of 24 breast cancer and immortalized mammary epithelial cell lines representing four of the six intrinsic molecular subtypes of breast cancer. We found that patterns of cell cycle checkpoint deregulation were associated with the intrinsic molecular subtype of breast cancer cell lines. Specifically, the luminal B and basal-like cell lines harbored two molecularly distinct Gap 2/mitosis checkpoint defects (impairment of the decatenation Gap 2 checkpoint and the spindle assembly checkpoint, respectively). All subtypes of breast cancer cell lines examined displayed aberrant DNA synthesis/Gap 2/mitosis progression and the basal-like and claudin-low cell lines exhibited increased percentages of chromatid cohesion defects. Furthermore, a decatenation Gap 2 checkpoint gene expression signature identified in the cell line panel correlated with clinical outcomes in breast cancer patients, suggesting that breast tumors may also harbor defects in decatenation Gap 2 checkpoint function. Taken together, these data imply that pharmacological targeting of signaling pathways driving these phenotypes may lead to the development of novel personalized treatment strategies for the latter two subtypes which currently lack targeted therapeutic options because of their triple negative breast cancer status.