Sequence analysis of cell-free DNA derived from cultured human bone osteosarcoma (143B) cells.

The true importance of cell-free DNA in human biology, together with the potential scale of its clinical utility, is tarnished by a lack of understanding of its composition and origin. In investigating the cell-free DNA present in the growth medium of cultured 143B cells, we previously demonstrated that the majority of cell-free DNA is neither a product of apoptosis nor necrosis. In the present study, we investigated the composition and origin of this cell-free DNA population using next-generation sequencing. We found that the cell-free DNA comprises mainly of repetitive DNA, including α-satellite DNA, mini satellites, and transposons that are currently active or exhibit the capacity to become reactivated. A significant portion of these cell-free DNA fragments originates from specific chromosomes, especially chromosomes 1 and 9. In healthy adult somatic cells, the centromeric and pericentromeric regions of these chromosomes are normally densely methylated. However, in many cancer types, these regions are preferentially hypomethylated. This can lead to double-stranded DNA breaks or it can directly impair the formation of proper kinetochore structures. This type of chromosomal instability is a precursor to the formation of nuclear anomalies, including lagging chromosomes and anaphase bridges. DNA fragments derived from these structures can recruit their own nuclear envelope and form secondary nuclear structures known as micronuclei, which can localize to the nuclear periphery and bud out from the membrane. We postulate that the majority of cell-free DNA present in the growth medium of cultured 143B cells originates from these micronuclei.

The diverse origins of circulating cell-free DNA in the human body: a critical re-evaluation of the literature.

Since the detection of cell-free DNA (cfDNA) in human plasma in 1948, it has been investigated as a non-invasive screening tool for many diseases, especially solid tumours and foetal genetic abnormalities. However, to date our lack of knowledge regarding the origin and purpose of cfDNA in a physiological environment has limited its use to more obvious diagnostics, neglecting, for example, its potential utility in the identification of predisposition to disease, earlier detection of cancers, and lifestyle-induced epigenetic changes. Moreover, the concept or mechanism of cfDNA could also have potential therapeutic uses such as in immuno- or gene therapy. This review presents an extensive compilation of the putative origins of cfDNA and then contrasts the contributions of cellular breakdown processes with active mechanisms for the release of cfDNA into the extracellular environment. The involvement of cfDNA derived from both cellular breakdown and active release in lateral information transfer is also discussed. We hope to encourage researchers to adopt a more holistic view of cfDNA research, taking into account all the biological pathways in which cfDNA is involved, and to give serious consideration to the integration of in vitro and in vivo research. We also wish to encourage researchers not to limit their focus to the apoptotic or necrotic fraction of cfDNA, but to investigate the intercellular messaging capabilities of the actively released fraction of cfDNA and to study the role of cfDNA in pathogenesis.

Valproic acid alters the content and function of the cell-free DNA released by hepatocellular carcinoma (HepG2) cells in vitro.

BACKGROUND: It has long been believed that cell-free DNA (cfDNA) actively released into circulation can serve as intercellular messengers, and their involvement in processes such as the bystander effect strongly support this. However, this intercellular messaging function of cfDNA may have clinical implications that have not yet been considered. METHODS: CfDNA was isolated from the growth medium of HepG2 cells treated with valproic acid (VPA). This cfDNA was then administered to untreated cells and cellular metabolic activity was measured. RESULTS: VPA altered the characteristics of cfDNA released by treated HepG2 cells in vitro. When administered to untreated cells, the cfDNA from cells treated with VPA resulted in the dose-dependent induction of glycolytic activity within 36 min of administration, but little to no alterations in oxidative phosphorylation. The glycolytic activity lasted for 4-6 h, whereas changes in subsequent cfDNA release and characteristics were found to remain persistent after two 24 h treatments. Fragmented genomic DNA from VPA-treated cells did not induce the effects observed for cfDNA obtained VPA-treated cells. CONCLUSIONS: It is possible for cfDNA to, under in vitro conditions, transfer pharmaceutically-induced effects to untreated recipient cells. Further investigation regarding this occurrence under in vivo conditions is, therefore, strongly encouraged. GENERAL SIGNIFICANCE: The intercellular messaging functions of cfDNA present in donated biological fluids has potential clinical implications that require urgent attention. These implications may, however, also have potential as new forms of treatment that can circumvent pharmacological barriers.

Cell-free DNA in a three-dimensional spheroid cell culture model: A preliminary study.

BACKGROUND: Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo state, may be of significant benefit for cfDNA research. METHODS: CfDNA was isolated from the growth medium of C3A spheroid cultures in rotating bioreactors during both normal growth and treatment with acetaminophen. Spheroid growth was monitored via planimetry, lactate dehydrogenase activity and glucose consumption and was related to isolated cfDNA characteristics. RESULTS: Changes in spheroid growth and stability were effectively mirrored by cfDNA characteristics. CfDNA characteristics correlated with that of previous two-dimensional (2D) cell culture and human plasma research. CONCLUSIONS: 3D spheroid cultures can serve as effective, simplified in vivo-simulating "closed-circuit" models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. BIOLOGICAL SIGNIFICANCE: 3D cell cultures can be used to translate "closed-circuit" in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth environment. Combining 3D culture and cfDNA research could, therefore, optimize both research fields.

Kinetic analysis, size profiling, and bioenergetic association of DNA released by selected cell lines in vitro.

Although circulating DNA (cirDNA) analysis shows great promise as a screening tool for a wide range of pathologies, numerous stumbling blocks hinder the rapid translation of research to clinical practice. This is related directly to the inherent complexity of the in vivo setting, wherein the influence of complex systems of interconnected cellular responses and putative DNA sources creates a seemingly arbitrary representation of the quantitative and qualitative properties of the cirDNA in the blood of any individual. Therefore, to evaluate the potential of in vitro cell cultures to circumvent the difficulties encountered in in vivo investigations, the purpose of this work was to elucidate the characteristics of the DNA released [cell-free DNA (cfDNA)] by eight different cell lines. This revealed three different forms of cfDNA release patterns and the presence of nucleosomal fragments as well as actively released forms of DNA, which are not only consistently observed in every tested cell line, but also in plasma samples. Correlations between cfDNA release and cellular origin, growth rate, and cancer status were also investigated by screening and comparing bioenergetics flux parameters. These results show statistically significant correlations between cfDNA levels and glycolysis, while no correlations between cfDNA levels and oxidative phosphorylation were observed. Furthermore, several correlations between growth rate, cancer status, and dependency on aerobic glycolysis were observed. Cell cultures can, therefore, successfully serve as closed-circuit models to either replace or be used in conjunction with biofluid samples, which will enable sharper focus on specific cell types or DNA origins.

Exposure to high levels of fumarate and succinate leads to apoptotic cytotoxicity and altered global DNA methylation profiles in vitro.

In the Krebs cycle, succinate is oxidized to fumarate by succinate dehydrogenase (SDH), followed by the conversion of fumarate to malate by fumarate hydratase (FH). In cells with defective SDH and FH, the Krebs cycle is congested, respiration impaired and fumarate and succinate accumulates. Several studies have indicated that the accumulation of these substrates are associated with cytotoxicity and oncogenesis. High levels of succinate and fumarate induce hypoxia inducible factor (HIF1A) hydroxylases, leading to the activation of oncogenic HIF pathways. However, the role of HIF as primary inducer of oncogenic change has been questioned, as other non-enzymatic mechanisms have been shown to interfere with cellular metabolism, cell signalling as well as disrupting protein function. Owing to the essential roles that SDH and FH play in cellular energy metabolism, and their associated tumor suppressor capacity, it is vital to understand the biochemical effects resulting from the accumulation of their associated metabolites. Therefore, in this study, we investigated the effect of high concentrations of succinate and fumarate exposure on cell viability, genome integrity and global DNA methylation using a human hepatocellular carcinoma (HepG2) cell culture model. It was found that relatively high concentrations of succinate and fumarate cause a loss of cell viability, which seems to be orchestrated through an apoptotic pathway. Cells exposed to high levels of succinate also presented with elevated caspase 3 and/or caspase 7 levels. In addition, elevated levels of fumarate lead to extensive DNA fragmentation, which may contribute pathophysiologically by inducing chromosomal instability, while succinate demonstrated lower genotoxicity. Furthermore, both succinate and fumarate altered the global DNA methylation patterns via significant DNA hypermethylation. Since numerous studies have reported correlations between aberrant DNA methylation and oncogenesis, hypermethylation may contribute to the oncogenesis observed in cells exposed to high concentrations of these metabolites.

An Enquiry Concerning the Characteristics of Cell-Free DNA Released by Cultured Cancer Cells.

Non-invasive screening that utilizes cell-free DNA (cfDNA) offers remarkable potential as a method for the early detection of genetic disorders and a wide variety of cancers. Unfortunately, one of the most prominent elements delaying the translation of cfDNA analyses to clinical practice is the lack of knowledge regarding its origin and composition. The elucidation of the origin of cfDNA is complicated by the apparently arbitrary variability of quantitative and qualitative characteristics of cfDNA in the blood of healthy as well as diseased individuals. These factors may contribute to false positive/negative results when applied to clinical pathology. Although many have acknowledged that this is a major problem, few have addressed it. We believe that many of the current difficulties encountered in in vivo cfDNA studies can be partially circumvented by in vitro models. The results obtained in this study indicate that the release of cfDNA from 143B cells is not a consequence of apoptosis, necrosis or a product of DNA replication, but primarily the result of actively released DNA, perhaps in association with a protein complex. Moreover, this study demonstrates the potential of in vitro cell culture models to obtain useful information about the phenomenon of cfDNA.

A Historical and Evolutionary Perspective on Circulating Nucleic Acids and Extracellular Vesicles: Circulating Nucleic Acids as Homeostatic Genetic Entities.

The quantitative and qualitative differences of circulating nucleic acids (cirNAs) between healthy and diseased individuals have motivated researchers to utilize these differences in the diagnosis and prognosis of various pathologies. The position maintained here is that reviewing the rather neglected early work associated with cirNAs and extracellular vesicles (EVs) is required to fully describe the nature of cirNAs. This review consists of an empirically up-to-date schematic summary of the major events that developed and integrated the concepts of heredity, genetic information and cirNAs. This reveals a clear pattern implicating cirNA as a homeostatic entity or messenger of genetic information. The schematic summary paints a picture of how cirNAs may serve as homeostatic genetic entities that promote synchrony of both adaptation and damage in tissues and organs depending on the source of the message.

A Quantitative Assessment of Cell-Free DNA Utilizing Several Housekeeping Genes: Measurements from Four Different Cell Lines.

Quantitative real-time PCR (qPCR) is regularly used to quantify cell-free nucleic acids (cfNAs) in order to identify biomarkers for various pathologies. However, studies have shown notable housekeeping gene expression variation between healthy and diseased tissues and treated versus untreated cell lines. The release of housekeeping genes by four cell lines was investigated and the housekeeping gene expression between cfNAs and mRNA of the cell lines was observed in order to elucidate their relationship.

Methodological Variables in the Analysis of Cell-Free DNA.

In recent years, cell-free DNA (cfDNA) analysis has received increasing amounts of attention as a potential non-invasive screening tool for the early detection of genetic aberrations and a wide variety of diseases, especially cancer. However, except for some prenatal tests and BEAMing, a technique used to detect mutations in various genes of cancer patients, cfDNA analysis is not yet routinely applied in clinical practice. Although some confusing biological factors inherent to the in vivo setting play a key part, it is becoming increasingly clear that this struggle is mainly due to the lack of an analytical consensus, especially as regards quantitative analyses of cfDNA. In order to use quantitative analysis of cfDNA with confidence, process optimization and standardization are crucial. In this work we aim to elucidate the most confounding variables of each preanalytical step that must be considered for process optimization and equivalence of procedures.

A historical and evolutionary perspective on the biological significance of circulating DNA and extracellular vesicles.

The discovery of quantitative and qualitative differences of the circulating DNA (cirDNA) between healthy and diseased individuals inclined researchers to investigate these molecules as potential biomarkers for non-invasive diagnosis and prognosis of various pathologies. However, except for some prenatal tests, cirDNA analyses have not been readily translated to clinical practice due to a lack of knowledge regarding its composition, function, and biological and evolutionary origins. We believe that, to fully grasp the nature of cirDNA and the extracellular vesicles (EVs) and protein complexes with which it is associated, it is necessary to probe the early and badly neglected work that contributed to the discovery and development of these concepts. Accordingly, this review consists of a schematic summary of the major events that developed and integrated the concepts of heredity, genetic information, cirDNA, EVs, and protein complexes. CirDNA enters target cells and provokes a myriad of gene regulatory effects associated with the messaging functions of various natures, disease progression, somatic genome variation, and transgenerational inheritance. This challenges the traditional views on each of the former topics. All of these discoveries can be traced directly back to the iconic works of Darwin, Lamarck, and their followers. The history of cirDNA that has been revisited here is rich in information that should be considered in clinical practice, when designing new experiments, and should be very useful for generating an empirically up-to-date view of cirDNA and EVs. Furthermore, we hope that it will invite many flights of speculation and stimulate further inquiry into its biological and evolutionary origins.

Reference gene selection for in vitro cell-free DNA analysis and gene expression profiling.

OBJECTIVES: (i) To optimize cell-free DNA (cfDNA) and mRNA quantification using eight housekeeping genes (HKGs), (ii) to determine if there is a difference in the occurrence of HKGs in the cfDNA and mRNA of normal cells and cancer cells, and (iii) to investigate whether there is some selectivity involved in the release of cfDNA. DESIGN AND METHODS: cfDNA was isolated directly from the growth medium of 3 cultured cancer cell lines and one non-malignant, primary cell line. At the same time interval, mRNA was isolated from these cells and cDNA was synthesized. CfDNA and cDNA were then amplified with real-time PCR utilizing eight different HKGs. RESULTS: For all cell lines tested, Beta-actin (ACTB) is the most appropriate HKG to use as a control for cfDNA and mRNA quantification. There was no clear difference in the occurrence of HKGs between cancer cells and healthy cells. Lastly, there is a consistent and distinct difference between the mRNA expression and cfDNA of all cell lines. CONCLUSIONS: This study reveals a new candidate HKG for a robust control in cfDNA analysis and gene expression profiling, and should be considered for optimal analysis. Furthermore, results indicate that cfDNA is selectively released from cells into culture medium.

Adjustments to the preanalytical phase of quantitative cell-free DNA analysis.

Evaluating the kinetics of cell-free DNA (cfDNA) in the blood of cancer patients could be a strong auxiliary component to the molecular characterization of cfDNA, but its potential clinical significance is obscured by the absence of an analytical consensus. To utilize quantitative cfDNA assessment with confidence, it is crucial that the preanalytical phase is standardized. In a previous publication, several preanalytical variables that may affect quantitative measurements of cfDNA were identified, and the most confounding variables were assessed further using the growth medium of cultured cancer cells as a source of cfDNA ("Cell-free DNA: Preanalytical variables" [1]). The data accompanying this report relates to these experiments, which includes numerous changes to the sample handling and isolation protocols, and can be used for the interpretation of these results and other similar experiments by different researchers.

Characterization of the cell-free DNA released by cultured cancer cells.

The most prominent factor that delays the translation of cell-free DNA (cfDNA) analyses to clinical practice is the lack of knowledge regarding its origin and composition. The elucidation of the former is complicated by the seemingly random fluctuation of quantitative and qualitative characteristics of cfDNA in the blood of healthy and diseased individuals. Besides methodological discrepancies, this could be ascribed to a web of cellular responses to various environmental cues and stressors. Since all cells release cfDNA, it follows that the cfDNA in the blood of cancer patients is not only representative of tumor derived DNA, but also of DNA released by healthy cells under different conditions. Additionally, cfDNA released by malignant cells is not necessarily just aberrant, but likely includes non-mutated chromosomal DNA fragments. This may cause false positive/negative results. Although many have acknowledged that this is a major problem, few have addressed it. We propose that many of the current stumbling blocks encountered in in vivo cfDNA studies can be partially circumvented by in vitro models. Accordingly, the purpose of this work was to evaluate the release of cfDNA from cultured cells and to gauge its potential use for elucidating the nature of cfDNA. Results suggest that the occurrence of cfDNA is not a consequence of apoptosis or necrosis, but primarily a result of actively secreted DNA, perhaps in association with a protein complex. This study demonstrates the potential of in vitro cell culture models to obtain useful information about the phenomenon of cfDNA.

Cell-free DNA: Preanalytical variables.

Since the discovery of cell-free DNA (cfDNA) in human blood, most studies have focused on diagnostic and prognostic uses of these markers for solid tumors. Except for some prenatal tests and BEAMing, cfDNA analysis has not yet been translated to clinical practice and routine application appears distant. This can be attributed to overlapping factors: (i) a lack of knowledge regarding the origin and function of cfDNA, (ii) insufficient molecular characterization, and (iii) the absence of an analytical consensus. In this review, we address the latter determinant and focus specifically on quantitative analysis of cfDNA. While the literature reports limited value for a single quantitative assessment, cfDNA kinetic assessment will be an essential component to qualitative characterization. In order to establish quantitative analysis for accurate kinetic assessments, process optimization and standardization are crucial. This report elucidates the most confounding variables of each preanalytic step that must be considered for optimal analysis.

Continuous adaptation through genetic communication - a putative role for cell-free DNA.

INTRODUCTION: The virtosome fraction of cell-free DNA, being newly synthesised, capable of horizontal gene transfer (HGT) and subsequent genomic incorporation and expression, may play crucial roles in the biological functions of higher organisms. In order to unlock the full potential of cell-free DNA as a biomarker, we need to fully understand its biological roles. AREAS COVERED: Metabolic DNA is possibly the precursor to the bulk of cell-free DNA, which is actively released as lipoprotein complexes. These released lipoprotein complexes are referred to as virtosomes. Research suggests that acquired characteristics, including physical and behavioural traits can be inherited in contrast to the neo-Darwinian dogma. We consider the virtosome fraction of cell-free DNA to be involved in this process. We suggest that at least one process of endoreplication is responsible for the synthesis of metabolic DNA and that this DNA follows apoptotic-like breakdown prior to release. EXPERT OPINION: If individual cellular genomes are capable of acquiring and donating the active parts of their genomes, selection of viable genetic information occurs not only through natural selection of whole organisms, but occurs continuously at a subgenomic level. The process of homeostatic adaptation in higher organisms is influenced by the characteristics of HGT.

Origin, translocation and destination of extracellular occurring DNA--a new paradigm in genetic behaviour.

The diagnostic value of extracellular occurring DNA (eoDNA) is limited by our lack of understanding its biological function. eoDNA exists in a number of forms, namely vesicle bound DNA (apoptotic bodies, micro particles, micro vesicles and exosomes), histone/DNA complexes or nucleosomes and virtosomes. These forms of DNA can also be categorized under the terms circulating DNA, cell free DNA, free DNA and extracellular DNA. The DNA can be released by means of form-specific mechanisms and seem to be governed by cell cycle phases and apoptosis. Active release is supported by evidence of energy dependent release mechanisms and various immunological- and messenger functions. Sequencing has shown that eoDNA sequences present in the nucleosome reflects traits and distribution of genome sequences and are regulated by ways of release and/or clearance. eoDNA enables the horizontal transfer of gene sequences from one cell to another, over various distances. The ability of eoDNA to partake in horizontal gene transfer makes it an important facet in the field of epigenetic variation. Clinical implementation of eoDNA diagnostics requires that all of the subgroups of eoDNA be properly investigated.

Characterisation of circulating DNA by parallel tagged sequencing on the 454 platform.

BACKGROUND: Fragments of genomic DNA that can be isolated from the blood and body fluids of vertebrates are also known as circulating DNA. This DNA has widely been investigated as a biomarker for cancer and other diseases but the origin and significance of circulating DNA have not been elucidated to date. METHODS: We used a parallel tagged sequencing method to sequence circulating DNA obtained from control individuals as well as cancer patients on the GSFLX sequencer (454 life sciences). RESULTS: Circulating DNA sequenced on one 16th of a picotiter plate produced approximately 3600 unique circulating DNA sequences which were distributed over the human genome and a higher frequency of mutations was observed in cancer patients compared to healthy controls. CONCLUSION: Circulating DNA represents genomic DNA in the blood of an individual, some sequence related differences might be evident between circulating DNA from cancer individuals and controls but distribution over the genome is similar.

Circulating DNA. Its origin and fluctuation.

Circulating DNA is present in the blood of all individuals, but it has been found that cancer patients and patients with a variety of other conditions have increased amounts of these circulating DNA fragments in their blood. Even though more than 30 years of research have been done on this subject, the origin of these nucleic acid molecules is still not clear. We present evidence that does not support the general notion that apoptosis or necrosis is the major source of circulating free DNA. Active release of free circulating DNA by living cells may be a plausible mechanism. Disturbance of the equilibrium between the release of DNA by living cells and the mechanisms used for clearing this DNA may play the main role in the appearance of increased amounts of circulating DNA in the blood of individuals with different ailments. Elucidating the origin and the mechanism that cells use to release free circulating DNA into the blood may enhance the diagnostic and prognostic value of these nucleic acid molecules.

A method for characterization of total circulating DNA.

Although much work has been done in the field of circulating DNA, no definitive information on sequencing data of total circulating DNA is available. Characterization of total circulating DNA by sequence analysis may give valuable information about the origin and function of these nucleic acid molecules. Circulating DNA was isolated from plasma of one healthy individual and one cancer patient with various methods and was cloned into a blunted cloning vector. Resulting colonies were sequenced and analyzed. The majority of the DNA that ligated into the vector was about 200 bp in length. Sequence analysis revealed that circulating DNA consists partly of currently uncharacterized human genomic sequences and when human repeats were masked it matched with sequences present in contigs containing known genes situated at various distances from the identified targets. In addition to the presence of large repeats, a variety of Alu repeat sequences were observed. Preliminary results showed that more Alu repeats are present in the plasma of normal individuals than in patient material. None of the gene sequences reported in the literature to be part of circulating DNA (e.g., P53, the Ras family, beta-globin, or beta-actin) was observed. Cloning and sequencing of free circulating DNA was successful and this first attempt on characterizing sequence data of free circulating DNA not only confirmed previous results, but also revealed a large variety of sequences. Further characterization of circulating DNA may be beneficial in diagnosis and prognosis and may also contribute to determining the source and function of circulating DNA.