Cell-free DNA as a biomarker in stroke: Current status, problems and perspectives.

There is currently no proposed stroke biomarker with consistent application in clinical practice. A number of studies have examined cell-free DNA (cfDNA), which circulates in biological fluids during stroke, as a potential biomarker of this disease. The data available suggest that dynamically-determined levels of blood cfDNA may provide new prognostic information for assessment of stroke severity and outcome. However, such an approach has its own difficulties and limitations. This review covers the potential role of cfDNA as a biomarker in stroke, and includes evidence from both animal models and clinical studies, protocols used to analyze cfDNA, and hypotheses on the origin of cfDNA.

Oxidized extracellular DNA as a stress signal that may modify response to anticancer therapy.

An increase in the levels of oxidation is a universal feature of genomic DNA of irradiated or aged or even malignant cells. In case of apoptotic death of stressed cells, oxidized DNA can be released in circulation (cfDNA). According to the results of the studies performed in vitro by our group and other researchers, the oxidized cfDNA serves as a biomarker for a stress and a stress signal that is transmitted from the "stressed" area i.e. irradiated cells or cells with deficient anti-oxidant defenses to distant (bystander) cells. In recipient cells, oxidized DNA stimulates biosynthesis of ROS that is followed up by an increase in the number of single strand and double strand breaks (SSBs and DSBs), and activation of DNA Damage Response (DDR) pathway. Effects of oxidized DNA are considered similar to that of irradiation. It seems that downstream effects of irradiation, in part, depend on the release of oxidized DNA fragments that mediate the effects in distant cells. The responses of normal and tumor cell to oxidized DNA may differ. It seems that tumor cells are more sensitive to oxidized DNA-dependent DNA damage, while developing pronounced adaptive response. This may suggest that in chemotherapy or irradiation-treated human body, the release of oxidized DNA from dying cancer cells may give a boost to remaining malignant cells by augmenting their survival and stress resistance. Further studies of the effects of oxidized DNA in both in vitro and in vivo systems are warranted.

Oxidized extracellular DNA as a stress signal in human cells.

The term "cell-free DNA" (cfDNA) was recently coined for DNA fragments from plasma/serum, while DNA present in in vitro cell culture media is known as extracellular DNA (ecDNA). Under oxidative stress conditions, the levels of oxidative modification of cellular DNA and the rate of cell death increase. Dying cells release their damaged DNA, thus, contributing oxidized DNA fragments to the pool of cfDNA/ecDNA. Oxidized cell-free DNA could serve as a stress signal that promotes irradiation-induced bystander effect. Evidence points to TLR9 as a possible candidate for oxidized DNA sensor. An exposure to oxidized ecDNA stimulates a synthesis of reactive oxygen species (ROS) that evokes an adaptive response that includes transposition of the homologous loci within the nucleus, polymerization and the formation of the stress fibers of the actin, as well as activation of the ribosomal gene expression, and nuclear translocation of NF-E2 related factor-2 (NRF2) that, in turn, mediates induction of phase II detoxifying and antioxidant enzymes. In conclusion, the oxidized DNA is a stress signal released in response to oxidative stress in the cultured cells and, possibly, in the human body; in particular, it might contribute to systemic abscopal effects of localized irradiation treatments.

Role of extracellular DNA oxidative modification in radiation induced bystander effects in human endotheliocytes.

The development of the bystander effect induced by low doses of irradiation in human umbilical vein endothelial cells (HUVECs) depends on extracellular DNA (ecDNA) signaling pathway. We found that the changes in the levels of ROS and NO production by human endothelial cells are components of the radiation induced bystander effect that can be registered at a low dose. We exposed HUVECs to X-ray radiation and studied effects of ecDNA(R) isolated from the culture media conditioned by the short-term incubation of irradiated cells on intact HUVECs. Effects of ecDNA(R) produced by irradiated cells on ROS and NO production in non-irradiated HUVECs are similar to bystander effect. These effects at least partially depend on TLR9 signaling. We compared the production of the nitric oxide and the ROS in human endothelial cells that were (1) irradiated at a low dose; (2) exposed to the ecDNA(R) extracted from the media conditioned by irradiated cells; and (3) exposed to human DNA oxidized in vitro. We found that the cellular responses to all three stimuli described above are essentially similar. We conclude that irradiation-related oxidation of the ecDNA is an important component of the ecDNA-mediated bystander effect.

An extracellular DNA mediated bystander effect produced from low dose irradiated endothelial cells.

The human umbilical vein endothelial cells culture was exposed to X-ray radiation in a low dose of 10cGy. The fragments of extracellular genomic DNA (ecDNA(R)) were isolated from the culture medium after the short-term incubation. A culture medium of unirradiated endothelial cells was then supplemented with ecDNA(R), followed by analysing the cells along the series of parameters (bystander effect). The exposed cells and bystander endotheliocytes showed similar response to low doses: approximation of the 1q12 loci of chromosome 1 and their transposition into the cellular nucleus, change in shape of the endotheliocytic nucleus, activation of the nucleolus organizing regions (NORs), actin polymerization, and an elevated level of DNA double-stranded breaks. Following blockade of TLR9 receptors with oligonucleotide-inhibitor or chloroquine in the bystander cells these effects - except of activation of NORs - on exposure to ecDNA(R) disappeared, with no bystander response thus observed. The presence of the radiation-induced apoptosis in the bystander effect being studied suggests a possibility for radiation-modified ecDNA fragments (i.e., stress signaling factors) to be released into the culture medium, whereas inhibition of TLR9 suggests the binding these ligands to the recipient cells. A similar DNA-signaling pathway in the bystander effect we previously described for human lymphocytes. Integrity of data makes it possible to suppose that a similar signaling mechanism which we demonstrated for lymphocytes (humoral system) might also be mediated in a monolayer culture of cells (cellular tissue) after the development of the bystander effect in them and transfer of stress signaling factors (ecDNA(R)) through the culture medium.

Oxidative stress as a significant factor for development of an adaptive response in irradiated and nonirradiated human lymphocytes after inducing the bystander effect by low-dose X-radiation.

X-radiation (10cGy) was shown to induce in human lymphocytes transposition of homologous chromosomes loci from the membrane towards the centre of the nucleus and activation of the chromosomal nucleolus-forming regions (NFRs). These effects are transmitted by means of extracellular DNA (ecDNA) fragments to nonirradiated cells (the so-called bystander effect, BE). We demonstrated that in the development of the BE an important role is played by oxidative stress (which is brought about by low radiation doses and ecDNA fragments of the culture medium of the irradiated cells), by an enzyme of apoptosis called caspase-3, and by DNA-binding receptors of the bystander cells, presumably TLR9. Proposed herein is a scheme of the development of an adaptive response and the BE on exposure to radiation. Ionizing radiation induces apoptosis of the radiosensitive fraction of cells due to the development of the "primary" oxidative stress (OS). DNA fragments of apoptotic cells are released into the intercellular space and interact with the DNA-binding receptors of the bystander cells. This interaction activates in lymphocytes signalling pathways associated with synthesis of the reactive oxygen species and nitrogen species, i.e., induces secondary oxidative stress accompanied by apoptosis of part of the cells, etc. Hence, single exposure to radiation may be followed by relatively long-lasting in the cellular population oxidative stress contributing to the development of an adaptive response. We thus believe that ecDNA of irradiated apoptotic lymphocytes is a significant factor of stress-signalling.

Extracellular DNA fragments.

During the development of the adaptive response, the pericentromeric loci of homologous chromosomes appear to move from the perimembrane sites of the cell nucleus and approach each other for a possible repair of double-stranded breaks of DNA in the process of homologous recombination. After exposure to X-ray radiation at an adapting dose of 10 cGy, transposition of the chromosomal pericentromeric loci and the accompanying activation of the chromosomal nucleolus-forming regions (NFRs) were observed in the irradiated lymphocytes, and were seen also in the intact bystander cells incubated in the growth medium of the exposed lymphocytes (the so-called bystander effect). From the culture medium of the irradiated and intact lymphocytes, we isolated DNA fragments that were introduced into the medium of nonirradiated cells in independent experiments. The bystander lymphocytes were found to demonstrate both transposition of the loci of homologous chromosomes and activation of the chromosomal NFRs, whereas after inoculation of the DNA fragments of the unirradiated cells, neither of the above effects was observed. Discussed herein are the characteristics of the factors revealed and possible pathways of stress signaling between the irradiated lymphocytes and the bystander cells.

Influence of plasma DNA on acid-base balance, blood gas measurement, and oxygen transport in health and stroke.

Hyperoxia and alkalemia, as a result of pulmonary hyperventilation and elevation of plasma DNA (pDNA), are seen during the first 24 h after ischemic stroke. In this study we have examined the correlation between pDNA and these blood parameters in health and stroke. Acid-base equilibrium, oxygen status, hemoglobin affinity to oxygen and concentration of pDNA in arterial blood were measured after the intravenous injection of homologous long-chain DNA to healthy rats and rats subjected to common carotid arterial occlusion. In addition the effect of adding homologous DNA to human and rat venous blood samples was studied in vitro. Hyperoxia, alkalemia, and an increase in hemoglobin affinity to oxygen were seen in rats with artificial stroke. A marked decrease in pulmonary hyperventilation and hemoglobin affinity to oxygen was observed after injection of homologous genomic DNA (10(-6) g/mL of blood). After the DNA injection, blood gas measurement and concentration of pDNA were correlated. Addition of DNA at a concentration of 10(-7) g/mL to venous blood samples in vitro increased oxygen saturation that disappeared when the dose of the DNA increased 10-fold. Thus, a change of pDNA concentration or size can alter acid-base equilibrium, oxygen status, and oxygen transport. These results may be important for a better understanding of the mechanisms of stroke and other diseases associated with the elevation of pDNA concentration, and they open the possibility of new therapeutic approaches.