|dc.description.abstract||Over the past 40 years, numerous proof-of-principle studies have intermittently demonstrated the translational potential of cfDNA as a non-invasive biomarker for the diagnostication, prognostication and therapy monitoring of a wide range of diseases, physiological conditions and other clinical scenarios. However, abstracting data in a research setting and applying it in medical practice proved to be more complicated than expected. It is commonly assumed that the development of comprehensive clinical cfDNA assays, along with the scope of the utility of cfDNA, is constrained mainly by a lack of an analytical consensus between research groups, and by the limits of current technologies. However, an increasing number of reports suggest that a lack of knowledge concerning the biological properties of cfDNA may be another substantial obstacle in the way of the rapid translation of research to medical practice.
Therefore, a major objective of this study was to develop a better understanding of the origin, structure, fluctuation and function of cfDNA in human biology. A review of the literature confirmed that the strain imposed on applied cfDNA research by methodological drawbacks, is indeed exacerbated by a poor understanding of the biological properties of cfDNA and by a lack of consideration thereof in clinical validation experiments. A multitude of intrinsic and extrinsic sources (e.g., apoptosis and necrosis) and causes (e.g., oxidative stress and bacterial turnover) can result in the presence of cfDNA in bio-fluids. Moreover, many of these sources and causes appear to be inextricably linked by a complex interplay of cellular and physiological interactions (e.g., endocrine signaling, metabolism, homeostasis), which are in turn influenced by a myriad of biological factors such as weight, fitness, health, diet, smoking, circadian oscillations and medicinal status, the nature of which can differ greatly between individuals of different age, gender and ethnicity, for example. The convergence of these factors result in a seemingly arbitrary presentation of both the quantitative and qualitative characteristics of cfDNA in the blood of an individual, and between individuals, at any instance, which severely complicates the characterization of cfDNA in vivo. In this regard, the development and utilization of alternative strategies for studying the biological properties of cfDNA is completely rationalized. Since two dimensional cell culture models are sequestered from many of the confounding elements inherent to the in vivo setting, it has the potential to overcome many of the obstacles associated with heterogeneous bio-fluid samples. However, despite its proven advantages in other domains of biological research, the application thereof in cfDNA research is largely lacking. Therefore, the most important aim of this study was to implement a cell culture model to investigate the biological properties of cfDNA.
Quantitative PCR and chip-based capillary electrophoresis in conjunction with flow cytometry revealed the presence of copious cfDNA fragments with a size of ~2000 bp in the growth media of 143B cells after 24 hours of incubation, which could not be correlated with apoptosis, necrosis or DNA replication. This indicated the involvement of some active release mechanism. Evaluation of different cancer and non-cancer cell lines suggested that this may be a common phenomenon. In an experiment intended for the optimization of cfDNA quantification and gene expression profiling, it was recognized that this ~2000 bp population is represented by different amounts of housekeeping genes, and that some housekeeping genes are absent in the cfDNA, despite being present in cellular mRNA. These studies suggested the intriguing possibility that there could be some intent and selectivity involved in the release of cfDNA.
Nucleotide sequencing of the actively released cfDNA revealed that the majority of this cfDNA consists of repetitive DNA (88 %), comprised largely of α-satellites and mini-satellites and the Alu, LINE1, ERV (K) class II, MaLR and TcMar-Tigger repetitive elements. A careful review of the literature indicated a strong correlation between the representation of these elements and their current transposition activity or their ability to become reactivated. Finally, local alignment analyses demonstrated that the majority of these sequences originate from the centromeres of chromosomes 1 and 16. Interestingly, it has been reported that the hypomethylation of DNA at the peri-centromeric regions of these two chromosomes leads to rearrangements, decondensation, and eventually chromosomal instability. Therefore, keeping in mind that hypomethylation is a hallmark of cancer cells, and that transposons can become reactivated by DNA demethylation, it was hypothesized that the demethylation of these regions in 143B cells leads to derepression and mobilization of transposons, followed by aberrant translocations and chromosomal instability. Based on the structural similarity between centromere protein B (CENP-B), a protein capable of inducing DNA breaks, and the transposase encoded by the Tigger DNA transposon, which are liable to activation by demethylation, both CENP-B and transposases may facilitate the excision of satellite DNA. Furthermore, considering the inextricably laced sequences of satellite DNA and transposons, it is likely that the presence of overrepresented transposons is a result of programmed DNA elimination.
Questions raised by these observations are whether satellite DNA and transposons are (i) deliberately released by cancer cells to perform specific functions in the extracellular environment, (ii) by-products of a normal cellular process and are incidentally biologically active, or (iii) biologically-inert byproducts. In this study it was not only demonstrated that certain repetitive element families are significantly overrepresented in the cfDNA released by 143B cells, but that specific members of each family are overrepresented, such as the L1P1 and HERVK9int subfamilies of LINE1 and ERV (K) class II, respectively.
The involvement of a single LINE1 element in the initiation of human colorectal cancer has recently been demonstrated, and the role of endogenous retroviruses in cancer is well documented. In keeping with this, numerous reports have described how cfDNA can be transported throughout the body, while other studies have demonstrated their capacity to enter target cells and alter their biology, with associated effects ranging from mutagenesis and oncogenesis to chemo-resistance and metastasis. Since the mechanisms involved in these phenomena are still unclear, satellite DNA and transposons may yet prove to be among the key effector molecules. Furthermore, a partial explanation for the phenomenon that cancer patients generally present with elevated levels of cfDNA can be derived from the observation made in this study that cultured cancer cells release notably more DNA than normal cells, and that this is related to their metabolism. This, together with the correlative relationship between the malignancy of cancer cells and rate of demethylation, suggests that the level of DNA release increases concomitantly with malignancy. Viewed in the light of the central theorem of the extended phenotype, in which the malignancy of cancer cells should maximize the survival of genetic instructions that promote malignant behavior, it stands to reason that cancer cells would up-regulate the mobilization and lateral transfer of transposons to neighboring cells with the purpose of transforming them. In line with this premise, it can be argued that the composition and function of the DNA released by normal cells will differ from cancer cells on a fundamental level, and it is also likely that the cfDNA from different cancer cells differ. Since it was demonstrated that normal cells also release DNA, these results not only implicate the active release of satellite DNA and transposons in detrimental effects, but also provide a potential mechanism for the transfer of satellite DNA and transposons between otherwise healthy somatic cells.
Taken together, the results and arguments presented in this thesis suggest that the commonly held assumption that apoptosis is the main origin, and most relevant fraction, of cfDNA in human blood may be incorrect, restrictive, and should be reconsidered. Further inquiry into the biological properties of actively released DNA will not only benefit applied research, but could also provide a new framework for a deeper understanding of molecular biology, pathology and the process of evolution. Furthermore, this study demonstrates the utility of in vitro cell culture models for studying the phenomenon of cfDNA, and as such also emphasizes the importance of consolidating basic and applied cfDNA research.||en_US