The Origin of Circulating Free DNA

Almost every report on circulating DNA identifies apoptosis or necrosis or both as the main source of free circulating DNA in serum and plasma. A hallmark of apoptosis is DNA degradation, in which chromosomal DNA is 1st cleaved into large fragments (50–300 kb) and subsequently into multiples of nucleosomal units (180–200 bp)(1). This ladder pattern is also visible after electrophoresis of circulating DNA and is frequently considered to be evidence that apoptosis may be the source of the observed DNA fragments in plasma(2)(3). It has been shown, however, that the characteristic ladder pattern can also be observed for actively released DNA(3). The contents of apoptotic cells are rapidly ingested by professional phagocytes or neighboring cells through mechanisms that are not fully understood(4), and the DNA is consequently completely digested by DNase II in lysosomes(1). Thus the possibility exists that DNA fragments released by apoptosis are removed before appearing in the circulation. If this engulfment of apoptotic bodies is impaired or cell death is increased enough to produce substantial amounts of circulating DNA, inflammation would definitely be a problem and autoimmunity would occur frequently in cancer and other conditions involving increased circulating DNA(1)(4). Radiotherapy, chemotherapy, and other cancer treatments cause cell death by apoptosis, and less circulating DNA is found in cancer patients after treatment than before treatment, possibly because of the inhibitory effect of treatment on the proliferation of cancer cells. Furthermore, in the early stages of cancer, when little cell death seems to occur, circulating DNA may already be present in higher than normal concentrations. As the cancer burden increases, so does the rate of cell death and the amount of proliferating cancer cells, with a concomitant increase in circulating DNA. In addition, many cancer cells are resistant to apoptosis, and to escape the immune system proliferating cells lose the ability to become apoptotic(2)(3), a process that also runs counter to the notion of apoptosis as the main mechanism for generating free DNA. Necrosis, on the other hand, produces large DNA fragments. Even with no cells dying, the DNA concentration in culture medium increases in proportion to the proliferation of cultured cancer cells. Human lymphocytes have also been observed to actively release double-stranded DNA into culture medium to a certain concentration(5), irrespective of incubation time. A similar observation was made in experiments with frog auricles: DNA was released to the same concentrations during successive transfer of auricles to fresh medium, purified frog DNA did not inhibit release of DNA, and damaged auricles did not yield more DNA into the medium(5). Therefore, quantities of released DNA are similar regardless of the proportion of dying cells(5), with the exception applicable for cancer cells, which can release more DNA than normal cells(2). Evidence for preferential release of DNA was found by comparing the proportion of Alu repeat sequences to β-globin in serum and lymphocytes(5). We thus conclude that apoptosis and necrosis are not the main source of circulating DNA, although they may contribute. DNA released by living cells is a viable alternative. The mechanism of DNA clearance from plasma is poorly understood. Increased amounts of circulating DNA in the blood of patients may reflect disturbance of the equilibrium between the release of DNA by living cells and the clearance of DNA. The low concentrations of circulating DNA in healthy individuals may reflect a lower rate of DNA release by cells or a rapid removal of DNA by the optimal functioning of clearance mechanisms, and when this equilibrium is disturbed, the amount of circulating DNA increases. Circulating DNA can be found in a variety of conditions, and although these conditions are unrelated, the presence of circulating nucleic acids is a common feature, and thus some kind of correlation ought to be found that may point to a common mechanism of origin. Although researchers have been studying the origin of circulating DNA for more than 30 years, the mechanism of release still has to be elucidated. A reasonable possibility is that more than one mechanism is involved; if so, the variables influencing the relative contributions and the interactions between the mechanisms must be understood for optimal utilization of this very valuable, minimally invasive biomarker. Thus more work needs to be done to determine the mechanism(s) of release and clearance as well as the significance of circulating DNA in the body. Grant/funding support: None declared. Financial disclosures: None declared. Acknowledgments: We thank Dr. Phiyani Lebea for proofreading the final manuscript.