Base Excision Repair: Mechanisms and Impact in Biology, Disease, and Medicine.

Base excision repair (BER) corrects forms of oxidative, deamination, alkylation, and abasic single-base damage that appear to have minimal effects on the helix. Since its discovery in 1974, the field has grown in several facets: mechanisms, biology and physiology, understanding deficiencies and human disease, and using BER genes as potential inhibitory targets to develop therapeutics. Within its segregation of short nucleotide (SN-) and long patch (LP-), there are currently six known global mechanisms, with emerging work in transcription- and replication-associated BER. Knockouts (KOs) of BER genes in mouse models showed that single glycosylase knockout had minimal phenotypic impact, but the effects were clearly seen in double knockouts. However, KOs of downstream enzymes showed critical impact on the health and survival of mice. BER gene deficiency contributes to cancer, inflammation, aging, and neurodegenerative disorders. Medicinal targets are being developed for single or combinatorial therapies, but only PARP and APE1 have yet to reach the clinical stage.

A new sub-pathway of long-patch base excision repair involving 5' gap formation.

Base excision repair (BER) is one of the most frequently used cellular DNA repair mechanisms and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a major sub-pathway of conventional long-patch BER that involves formation of a 9-nucleotide gap 5' to the lesion. This new sub-pathway is mediated by RECQ1 DNA helicase and ERCC1-XPF endonuclease in cooperation with PARP1 poly(ADP-ribose) polymerase and RPA The novel gap formation step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates PARP1 auto-(ADP-ribosyl)ation and the choice between long-patch and single-nucleotide BER, thereby modulating cellular sensitivity to DNA damage. Based on these results, we propose a revised model of long-patch BER and a new key regulation point for pathway choice in BER.

Establishment of ornithine transcarbamylase deficiency-derived primary human hepatocyte with hepatic functions.

A long-term hepatocyte culture maintaining liver-specific functions is very essential for both basic research and the development of bioartificial liver devices in clinical application. However, primary hepatocytes rapidly lose their proliferation and hepatic functions over a few days in culture. This work is to establish an ornithine transcarbamylase deficiency (OTCD) patient-derived primary human hepatocyte (OTCD-PHH) culture with hepatic functions for providing an in vitro cell model. Liver tissue from an infant with OTCD was dispersed into single cells. The cells were cultured using conditional reprogramming. To characterize the cells, we assessed activities and mRNA expression of CYP3A4, 1A1, 2C9, as well as albumin and urea secretion. We found that the OTCD-PHH can be subpassaged for more than 15 passages. The cells do not express mRNA of fibroblast-specific maker, whereas they highly express markers of epithelial cells and hepatocytes. In addition, the OTCD-PHH retain native CYP3A4, 1A1, 2C9 activities and albumin secretion function at early passages. The OTCD-PHH at passages 2, 6, 9 and 13 have identical DNA fingerprint as the original tissue. Furthermore, under 3D culture environment, low urea production and hepatocyte marker staining of the OTCD-PHH were detected. The established OTCD-PHH maintain liver-specific functions at early passages and can be long-term cultured in vitro. We believe the established long-term OTCD-PHH culture is highly relevant to study liver diseases, particularly in infants with OTCD.

Germ line variants of human N-methylpurine DNA glycosylase show impaired DNA repair activity and facilitate 1,N6-ethenoadenine-induced mutations.

Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N(6)-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (KD) of hMPG toward 1,N(6)-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.

RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin.

RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in replication restart mechanisms. Cellular phenotypes of RECQ1 deficiency are indicative of aberrant repair of stalled replication forks, but the molecular functions of RECQ1, the most abundant of the five known human RecQ homologues, have remained poorly understood. We show that RECQ1 associates with FEN-1 (flap endonuclease-1) in nuclear extracts and exhibits direct protein interaction in vitro. Recombinant RECQ1 significantly stimulated FEN-1 endonucleolytic cleavage of 5'-flap DNA substrates containing non-telomeric or telomeric repeat sequence. RECQ1 and FEN-1 were constitutively present at telomeres and their binding to the telomeric chromatin was enhanced following DNA damage. Telomere residence of FEN-1 was dependent on RECQ1 since depletion of RECQ1 reduced FEN-1 binding to telomeres in unperturbed cycling cells. Our results confirm a conserved collaboration of human RecQ helicases with FEN-1 and suggest both overlapping and specialized roles of RECQ1 in the processing of DNA structure intermediates proposed to arise during replication, repair and recombination.

Slow repair of lipid peroxidation-induced DNA damage at p53 mutation hotspots in human cells caused by low turnover of a DNA glycosylase.

Repair of oxidative stress- and inflammation-induced DNA lesions by the base excision repair (BER) pathway prevents mutation, a form of genomic instability which is often observed in cancer as 'mutation hotspots'. This suggests that some sequences have inherent mutability, possibly due to sequence-related differences in repair. This study has explored intrinsic mutability as a consequence of sequence-specific repair of lipid peroxidation-induced DNA adduct, 1, N(6)-ethenoadenine (εA). For the first time, we observed significant delay in repair of ϵA at mutation hotspots in the tumor suppressor gene p53 compared to non-hotspots in live human hepatocytes and endothelial cells using an in-cell real time PCR-based method. In-cell and in vitro mechanism studies revealed that this delay in repair was due to inefficient turnover of N-methylpurine-DNA glycosylase (MPG), which initiates BER of εA. We determined that the product dissociation rate of MPG at the hotspot codons was ≈5-12-fold lower than the non-hotspots, suggesting a previously unknown mechanism for slower repair at mutation hotspots and implicating sequence-related variability of DNA repair efficiency to be responsible for mutation hotspot signatures.

Disruptive chemicals, senescence and immortality.

Carcinogenesis is thought to be a multistep process, with clonal evolution playing a central role in the process. Clonal evolution involves the repeated 'selection and succession' of rare variant cells that acquire a growth advantage over the remaining cell population through the acquisition of 'driver mutations' enabling a selective advantage in a particular micro-environment. Clonal selection is the driving force behind tumorigenesis and possesses three basic requirements: (i) effective competitive proliferation of the variant clone when compared with its neighboring cells, (ii) acquisition of an indefinite capacity for self-renewal, and (iii) establishment of sufficiently high levels of genetic and epigenetic variability to permit the emergence of rare variants. However, several questions regarding the process of clonal evolution remain. Which cellular processes initiate carcinogenesis in the first place? To what extent are environmental carcinogens responsible for the initiation of clonal evolution? What are the roles of genotoxic and non-genotoxic carcinogens in carcinogenesis? What are the underlying mechanisms responsible for chemical carcinogen-induced cellular immortality? Here, we explore the possible mechanisms of cellular immortalization, the contribution of immortalization to tumorigenesis and the mechanisms by which chemical carcinogens may contribute to these processes.

Metabolic reprogramming and dysregulated metabolism: cause, consequence and/or enabler of environmental carcinogenesis?

Environmental contributions to cancer development are widely accepted, but only a fraction of all pertinent exposures have probably been identified. Traditional toxicological approaches to the problem have largely focused on the effects of individual agents at singular endpoints. As such, they have incompletely addressed both the pro-carcinogenic contributions of environmentally relevant low-dose chemical mixtures and the fact that exposures can influence multiple cancer-associated endpoints over varying timescales. Of these endpoints, dysregulated metabolism is one of the most common and recognizable features of cancer, but its specific roles in exposure-associated cancer development remain poorly understood. Most studies have focused on discrete aspects of cancer metabolism and have incompletely considered both its dynamic integrated nature and the complex controlling influences of substrate availability, external trophic signals and environmental conditions. Emerging high throughput approaches to environmental risk assessment also do not directly address the metabolic causes or consequences of changes in gene expression. As such, there is a compelling need to establish common or complementary frameworks for further exploration that experimentally and conceptually consider the gestalt of cancer metabolism and its causal relationships to both carcinogenesis and the development of other cancer hallmarks. A literature review to identify environmentally relevant exposures unambiguously linked to both cancer development and dysregulated metabolism suggests major gaps in our understanding of exposure-associated carcinogenesis and metabolic reprogramming. Although limited evidence exists to support primary causal roles for metabolism in carcinogenesis, the universality of altered cancer metabolism underscores its fundamental biological importance, and multiple pleiomorphic, even dichotomous, roles for metabolism in promoting, antagonizing or otherwise enabling the development and selection of cancer are suggested.

Environmental immune disruptors, inflammation and cancer risk.

An emerging area in environmental toxicology is the role that chemicals and chemical mixtures have on the cells of the human immune system. This is an important area of research that has been most widely pursued in relation to autoimmune diseases and allergy/asthma as opposed to cancer causation. This is despite the well-recognized role that innate and adaptive immunity play as essential factors in tumorigenesis. Here, we review the role that the innate immune cells of inflammatory responses play in tumorigenesis. Focus is placed on the molecules and pathways that have been mechanistically linked with tumor-associated inflammation. Within the context of chemically induced disturbances in immune function as co-factors in carcinogenesis, the evidence linking environmental toxicant exposures with perturbation in the balance between pro- and anti-inflammatory responses is reviewed. Reported effects of bisphenol A, atrazine, phthalates and other common toxicants on molecular and cellular targets involved in tumor-associated inflammation (e.g. cyclooxygenase/prostaglandin E2, nuclear factor kappa B, nitric oxide synthesis, cytokines and chemokines) are presented as example chemically mediated target molecule perturbations relevant to cancer. Commentary on areas of additional research including the need for innovation and integration of systems biology approaches to the study of environmental exposures and cancer causation are presented.

Chemical compounds from anthropogenic environment and immune evasion mechanisms: potential interactions.

An increasing number of studies suggest an important role of host immunity as a barrier to tumor formation and progression. Complex mechanisms and multiple pathways are involved in evading innate and adaptive immune responses, with a broad spectrum of chemicals displaying the potential to adversely influence immunosurveillance. The evaluation of the cumulative effects of low-dose exposures from the occupational and natural environment, especially if multiple chemicals target the same gene(s) or pathway(s), is a challenge. We reviewed common environmental chemicals and discussed their potential effects on immunosurveillance. Our overarching objective was to review related signaling pathways influencing immune surveillance such as the pathways involving PI3K/Akt, chemokines, TGF-ß, FAK, IGF-1, HIF-1α, IL-6, IL-1α, CTLA-4 and PD-1/PDL-1 could individually or collectively impact immunosurveillance. A number of chemicals that are common in the anthropogenic environment such as fungicides (maneb, fluoxastrobin and pyroclostrobin), herbicides (atrazine), insecticides (pyridaben and azamethiphos), the components of personal care products (triclosan and bisphenol A) and diethylhexylphthalate with pathways critical to tumor immunosurveillance. At this time, these chemicals are not recognized as human carcinogens; however, it is known that they these chemicalscan simultaneously persist in the environment and appear to have some potential interfere with the host immune response, therefore potentially contributing to promotion interacting with of immune evasion mechanisms, and promoting subsequent tumor growth and progression.

The effect of environmental chemicals on the tumor microenvironment.

Potentially carcinogenic compounds may cause cancer through direct DNA damage or through indirect cellular or physiological effects. To study possible carcinogens, the fields of endocrinology, genetics, epigenetics, medicine, environmental health, toxicology, pharmacology and oncology must be considered. Disruptive chemicals may also contribute to multiple stages of tumor development through effects on the tumor microenvironment. In turn, the tumor microenvironment consists of a complex interaction among blood vessels that feed the tumor, the extracellular matrix that provides structural and biochemical support, signaling molecules that send messages and soluble factors such as cytokines. The tumor microenvironment also consists of many host cellular effectors including multipotent stromal cells/mesenchymal stem cells, fibroblasts, endothelial cell precursors, antigen-presenting cells, lymphocytes and innate immune cells. Carcinogens can influence the tumor microenvironment through effects on epithelial cells, the most common origin of cancer, as well as on stromal cells, extracellular matrix components and immune cells. Here, we review how environmental exposures can perturb the tumor microenvironment. We suggest a role for disrupting chemicals such as nickel chloride, Bisphenol A, butyltins, methylmercury and paraquat as well as more traditional carcinogens, such as radiation, and pharmaceuticals, such as diabetes medications, in the disruption of the tumor microenvironment. Further studies interrogating the role of chemicals and their mixtures in dose-dependent effects on the tumor microenvironment could have important general mechanistic implications for the etiology and prevention of tumorigenesis.

Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression.

As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: focus on the cancer hallmark of tumor angiogenesis.

One of the important 'hallmarks' of cancer is angiogenesis, which is the process of formation of new blood vessels that are necessary for tumor expansion, invasion and metastasis. Under normal physiological conditions, angiogenesis is well balanced and controlled by endogenous proangiogenic factors and antiangiogenic factors. However, factors produced by cancer cells, cancer stem cells and other cell types in the tumor stroma can disrupt the balance so that the tumor microenvironment favors tumor angiogenesis. These factors include vascular endothelial growth factor, endothelial tissue factor and other membrane bound receptors that mediate multiple intracellular signaling pathways that contribute to tumor angiogenesis. Though environmental exposures to certain chemicals have been found to initiate and promote tumor development, the role of these exposures (particularly to low doses of multiple substances), is largely unknown in relation to tumor angiogenesis. This review summarizes the evidence of the role of environmental chemical bioactivity and exposure in tumor angiogenesis and carcinogenesis. We identify a number of ubiquitous (prototypical) chemicals with disruptive potential that may warrant further investigation given their selectivity for high-throughput screening assay targets associated with proangiogenic pathways. We also consider the cross-hallmark relationships of a number of important angiogenic pathway targets with other cancer hallmarks and we make recommendations for future research. Understanding of the role of low-dose exposure of chemicals with disruptive potential could help us refine our approach to cancer risk assessment, and may ultimately aid in preventing cancer by reducing or eliminating exposures to synergistic mixtures of chemicals with carcinogenic potential.

The impact of low-dose carcinogens and environmental disruptors on tissue invasion and metastasis.

The purpose of this review is to stimulate new ideas regarding low-dose environmental mixtures and carcinogens and their potential to promote invasion and metastasis. Whereas a number of chapters in this review are devoted to the role of low-dose environmental mixtures and carcinogens in the promotion of invasion and metastasis in specific tumors such as breast and prostate, the overarching theme is the role of low-dose carcinogens in the progression of cancer stem cells. It is becoming clearer that cancer stem cells in a tumor are the ones that assume invasive properties and colonize distant organs. Therefore, low-dose contaminants that trigger epithelial-mesenchymal transition, for example, in these cells are of particular interest in this review. This we hope will lead to the collaboration between scientists who have dedicated their professional life to the study of carcinogens and those whose interests are exclusively in the arena of tissue invasion and metastasis.

The potential for chemical mixtures from the environment to enable the cancer hallmark of sustained proliferative signalling.

The aim of this work is to review current knowledge relating the established cancer hallmark, sustained cell proliferation to the existence of chemicals present as low dose mixtures in the environment. Normal cell proliferation is under tight control, i.e. cells respond to a signal to proliferate, and although most cells continue to proliferate into adult life, the multiplication ceases once the stimulatory signal disappears or if the cells are exposed to growth inhibitory signals. Under such circumstances, normal cells remain quiescent until they are stimulated to resume further proliferation. In contrast, tumour cells are unable to halt proliferation, either when subjected to growth inhibitory signals or in the absence of growth stimulatory signals. Environmental chemicals with carcinogenic potential may cause sustained cell proliferation by interfering with some cell proliferation control mechanisms committing cells to an indefinite proliferative span.

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Disruptive environmental chemicals and cellular mechanisms that confer resistance to cell death.

Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis.

Hepatitis C virus Genotype 1a core gene nucleotide patterns associated with hepatocellular carcinoma risk.

Specific sequence changes in codons 70 and 91 of the hepatitis C virus genotype 1b (HCV GT1b) core gene have been associated with increased risk of hepatocellular carcinoma (HCC). Essentially all previous studies were conducted in Asian populations with a wide range of liver disease, and none were conducted specifically in GT1a-infected individuals. We conducted a pilot study in a multiethnic population in the USA with HCV-related cirrhosis to determine if this association extended to GT1a-infected individuals and to determine if other sequence changes in the HCV core gene were associated with HCC risk. HCV core gene sequences from sera of 90 GT1 HCV carriers with cirrhosis (42 with HCC) were analysed using standard RT-PCR-based procedures. Nucleotide sequence data were compared with reference sequences available from GenBank. The frequency of sequence changes in codon 91 was not statistically different between HCC (7/19) and non-HCC (11/22) GT1b carriers. In GT1a carriers, sequence changes in codon 91 were observed less often than in GT1b carriers but were not observed in non-HCC subjects (4/23 vs 0/26, P = 0.03, Fisher's exact test). Sequence changes in codon 70 were not distributed differently between HCC and non-HCC GT1a and 1b carriers. Most importantly, for GT1a carriers, a panel of specific nucleotide changes in other codons was collectively present in all subjects with HCC, but not in any of the non-HCC patients. The utility of this test panel for early detection of HCC in GT1a-infected individuals needs to be assessed in larger populations, including longitudinal studies.

Naturally occurring polyphenol, morin hydrate, inhibits enzymatic activity of N-methylpurine DNA glycosylase, a DNA repair enzyme with various roles in human disease.

Interest in the mechanisms of DNA repair pathways, including the base excision repair (BER) pathway specifically, has heightened since these pathways have been shown to modulate important aspects of human disease. Modulation of the expression or activity of a particular BER enzyme, N-methylpurine DNA glycosylase (MPG), has been demonstrated to play a role in carcinogenesis and resistance to chemotherapy as well as neurodegenerative diseases, which has intensified the focus on studying MPG-related mechanisms of repair. A specific small molecule inhibitor for MPG activity would be a valuable biochemical tool for understanding these repair mechanisms. By screening several small molecule chemical libraries, we identified a natural polyphenolic compound, morin hydrate, which inhibits MPG activity specifically (IC50=2.6µM). Detailed mechanism analysis showed that morin hydrate inhibited substrate DNA binding of MPG, and eventually the enzymatic activity of MPG. Computational docking studies with an x-ray derived MPG structure as well as comparison studies with other structurally-related flavonoids offer a rationale for the inhibitory activity of morin hydrate observed. The results of this study suggest that the morin hydrate could be an effective tool for studying MPG function and it is possible that morin hydrate and its derivatives could be utilized in future studies focused on the role of MPG in human disease.

Redox regulation of apurinic/apyrimidinic endonuclease 1 activity in Long-Evans Cinnamon rats during spontaneous hepatitis.

The Long-Evans Cinnamon (LEC) rat is an animal model for Wilson's disease. This animal is genetically predisposed to copper accumulation in the liver, increased oxidative stress, accumulation of DNA damage, and the spontaneous development of hepatocellular carcinoma. Thus, this animal model is useful for studying the relationship of endogenous DNA damage to spontaneous carcinogenesis. In this study, we have investigated the apurinic/apyrimidinic endonuclease 1 (APE1)-mediated excision repair of endogenous DNA damage, apurinic/apyrimidinic (AP)-sites, which is highly mutagenic and implicated in human cancer. We found that the activity was reduced in the liver extracts from the acute hepatitis period of LEC rats as compared with extracts from the age-matched Long-Evans Agouti rats. The acute hepatitis period had also a heightened oxidative stress condition as assessed by an increase in oxidized glutathione level and loss of enzyme activity of glyceraldehyde 3-phosphate dehydrogenase, a key redox-sensitive protein in cells. Interestingly, the activity reduction was not due to changes in protein expression but apparently by reversible protein oxidation as the addition of reducing agents to extracts of the liver from acute hepatitis period reactivated APE1 activity and thus, confirmed the oxidation-mediated loss of APE1 activity under increased oxidative stress. These findings show for the first time in an animal model that the repair mechanism of AP-sites is impaired by increased oxidative stress in acute hepatitis via redox regulation which contributed to the increased accumulation of mutagenic AP-sites in liver DNA.