Effect of Low-Dose Ionizing Radiation on the Expression of Mitochondria-Related Genes in Human Mesenchymal Stem Cells.

The concept of hormesis describes a phenomenon of adaptive response to low-dose ionizing radiation (LDIR). Similarly, the concept of mitohormesis states that the adaptive program in mitochondria is activated in response to minor stress effects. The mechanisms of hormesis effects are not clear, but it is assumed that they can be mediated by reactive oxygen species. Here, we studied effects of LDIR on mitochondria in mesenchymal stem cells. We have found that X-ray radiation at a dose of 10 cGy as well as oxidized fragments of cell-free DNA (cfDNA) at a concentration of 50 ng/mL resulted in an increased expression of a large number of genes regulating the function of the mitochondrial respiratory chain complexes in human mesenchymal stem cells (MSC). Several genes remained upregulated within hours after the exposure. Both X-ray radiation and oxidized cfDNA resulted in upregulation of FIS1 and MFN1 genes, which regulated fusion and fission of mitochondria, within 3-24 h after the exposure. Three hours after the exposure, the number of copies of mitochondrial DNA in cells had increased. These findings support the hypothesis that assumes oxidized cell-free DNA as a mediator of MSC response to low doses of radiation.

Extracellular DNA Containing (dG)n Motifs Penetrates into MCF7 Breast Cancer Cells, Induces the Adaptive Response, and Can Be Expressed.

BACKGROUND: Oxidized human DNA or plasmid DNAs containing human ribosomal genes can easily penetrate into the breast cancer cells MCF7 and stimulate the adaptive response induction. Plasmid DNA containing a CMV promoter, gene EGFP, and the insertion of the human ribosomal genes can be expressed. A hypothesis is proposed: these features of the ribosomal DNA are due to the presence of dGn motifs that are prone to oxidize. METHODS: Cells of MCF7 line were cultured with plasmids which contained a CMV promoter and gene of fluorescent protein EGFP. Genetic construction pEGFP-Gn contains pEGFP vector and a small insertion with dG11 and dG13 motifs that are inclined to oxidation. The accumulation of pEGFP and pEGFP-Gn in MCF7 (qPCR), the levels of ROS in the cells, the content of 8-oxodG in plasmids and cellular DNA (flow cytometry, immunoassay, and fluorescent microscopy), the expression of NOX4 and EGFP, the localization of NOX4 and EGFP in MCF7 (qPCR, flow cytometry, and fluorescent microscopy), and the levels of the cell DNA damage (comet assay) were analyzed. RESULTS: (dG)n insertions in the plasmid pEGFP increase the levels of ROS, the cell DNA oxidation and DNA damage, and the level of transfection of plasmid into the MCF7 cells. NOX4 participates in the oxidation of pEGFP-Gn and pEGFP. The expression of EGFP gene in MCF7 is significantly increased in case of pEGFP-Gn. Stimulation of ROS synthesis (H2O2 40 µM or 10 cGy IR) increases the level of expression of EGFP. CONCLUSIONS: GC-rich DNA fragments containing dGn motifs that are inclined to oxidation penetrate into MCF7 cancer cells, stimulate the adaptive response, and can be expressed. This property of GC-rich cell-free DNA should be considered and/or could potentially be used in therapy of tumors.

Oxidized Cell-Free DNA Is a Factor of Stress Signaling in Radiation-Induced Bystander Effects in Different Types of Human Cells.

In pathology or under damaging conditions, the properties of cell-free DNA (cfDNA) change. An example of such change is GC enrichment, which drastically alters the biological properties of cfDNA. GC-rich cfDNA is a factor of stress signaling, whereas genomic cfDNA is biologically inactive. GC-rich cfDNA stimulates TLR9-MyD88-NF-κB signaling cascade, leading to an increase in proinflammatory cytokine levels in the organism. In addition, GC-rich DNA is prone to oxidation and oxidized cfDNA can stimulate secondary oxidative stress. This article is a review of works dedicated to the investigation of a low-dose ionizing radiation effect, a bystander effect, and the role of cfDNA in both of these processes.

Copy Number Variation of Human Satellite III (1q12) With Aging.

Introduction: Human satellite DNA is organized in long arrays in peri/centromeric heterochromatin. There is little information about satellite copy number variants (CNVs) in aging and replicative cell senescence (RS). Materials and Methods: Biotinylated pUC1.77 probe was used for the satellite III (f-SatIII) quantitation in leukocyte DNA by the non-radioactive quantitative hybridization for 557 subjects between 2 and 91 years old. The effect of RS and genotoxic stress (GS, 4 or 6 µM of K2CrO4) on the f-SatIII CNV was studied on the cultured human skin fibroblast (HSF) lines of five subjects. Results: f-SatIII in leukocyte and HSFs varies between 5.7 and 40 pg/ng of DNA. During RS, the f-SatIII content in HSFs increased. During GS, HSFs may increase or decrease f-SatIII content. Cells with low f-SatIII content have the greatest proliferative potential. F-SatIII CNVs in different individuals belonging to the different generations depend on year of their birth. Children (born in 2005-2015 years) differed significantly from the other age groups by low content and low coefficient of variation of f-SatIII. In the individuals born in 1912-1925 and living in unfavorable social conditions (FWW, the Revolution and the Russian Civil War, SWW), there is a significant disproportion in the content of f-SatIII. The coefficient of variation reaches the maximum values than in individuals born in the period from 1926 to 1975. In the group of people born in 1990-2000 (Chernobyl disaster, the collapse of the Soviet Union, and a sharp decline in the population living standard), again, there is a significant disproportion of individuals in the content of f-SatIII. A similar disproportion was observed in the analysis of a group of individuals born in 1926-1975 who in their youth worked for a long time in high-radioactive environment. Conclusion: In generations that were born and who lived in childhood in a period of severe social perturbations or in conditions of environmental pollution, we found a significant increase in leukocyte DNA f-SatIII variability. It is hypothesized that the change of the f-SatIII content in the blood cells reflects the body response to stress of different nature and intensity.

Ribosomal DNA as DAMPs Signal for MCF7 Cancer Cells.

Introduction: The cell free ribosomal DNA (cf-rDNA) is accrued in the total pool of cell free DNA (cfDNA) in some non-cancer diseases and demonstrates DAMPs characteristics. The major research questions: (1) How does cell free rDNA content change in breast cancer; (2) What type of response in the MCF7 breast cancer cells is caused by cf-rDNA; and (3) What type of DNA sensors (TLR9 or AIM2) is stimulated in MCF7 in response to the action of cf-rDNA? Materials and Methods: CfDNA and gDNA were isolated from the blood plasma and the cells derived from 38 breast cancer patients and 20 healthy female controls. The rDNA content in DNA was determined using non-radioactive quantitative hybridization. In order to explore the rDNA influence on MCF7 breast cancer cells, the model constructs (GC-DNAs) were applied: pBR322-rDNA plasmid (rDNA inset 5836 bp long) and pBR322 vector. ROS generation, DNA damage, cell cycle, expression of TLR9, AIM2, NF-kB, STAT3, and RNA for 44 genes affecting the cancer cell viability were evaluated. The methods used: RT-qPCR, fluorescent microscopy, immunoassay, flow cytometry, and siRNA technology. Results: The ratio R = cf-rDNA/g-rDNA for the cases was higher than for the controls (median 3.4 vs. 0.8, p < 10-8). In MCF7, GC-DNAs induce a ROS burst, DNA damage response, and augmentation of NF-kB and STAT3 activity. The number of the apoptotic cells decreases, while the number of cells with an instable genome (G2/M- arrest, micronuclei) increase. Expression of anti-apoptotic genes (BCL2, BCL2A1, BCL2L1, BIRC3, MDM2) is elevated, while expression of pro-apoptotic genes (BAX, BID, BAD, PMAIP1, BBC3) is lowered. The cells response for pBR322-rDNA is much more intense and develops much faster, than response for pBR322, and is realized through activation of TLR9- MyD88 - NF-kB- signaling. This difference in response speed is owing to the heightened oxidability of pBR322-rDNA and better ability to penetrate the cell. Induction of TLR9 expression in MCF7 is followed by blocking AIM2 expression. Conclusion: (1) Ribosomal DNA accumulates in cfDNA of breast cancer patients; (2) Cell free rDNA induce DNA damage response and stimulates cells survival, including cells with an instable genome; (3) Cell free rDNA triggers TLR9- MyD88- NF-kB- signaling, with significantly repressing the expression of AIM2.

Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell.

OBJECTIVE: Easily oxidizable GC-rich DNA (GC-DNA) fragments accumulate in the cell-free DNA (cfDNA) of patients with various diseases. The human oxidized DNA penetrates the MCF7 breast cancer cells and significantly changes their physiology. It can be assumed that readily oxidizable GC-DNA fragments can penetrate the cancer cells and be expressed. METHODS: MCF7 cells were cultured in the presence of two types of GC-DNA probes: (1) vectors pBR322 and pEGFP and (2) plasmids carrying inserted human rDNA (pBR322-rDNA and pEGFP-rDNA). pEGFP and pEGFP-rDNA contained a CMV promoter and a fluorescent protein gene EGFP. ROS generation rate, accumulation of the DNA probes in MCF7, 8-oxodG content, expression of EGFP and NOX4, and localization of EGFP, NOX4, and 8-oxodG in MCF7 were explored. The applied methods were qPCR, fluorescent microscopy (FM), immunoassay, and flow cytometry (FCA). RESULTS: When GC-DNA is added to the cell culture medium, it interacts with the cell surface. At the site of GC-DNA contact with the cell, NOX4 is expressed, and ROS level increases. The ROS oxidize the GC-DNA. When using the plasmids pEGFP and pEGFP-rDNA, an increase in the amount of the DNA EGFP, RNA EGFP, and EGFP proteins was detected in the cells. These facts suggest that GC-DNA penetrates the cells and the EGFP gene is expressed. Insertions of the rDNA significantly increase the GC-DNA oxidation degree as well as the rate of plasmid transfection into the cells and the EGFP expression level. In the nucleus, the oxidized GC-rDNA fragments, but not the vectors, are localized within the nucleolus. CONCLUSIONS: GC-rich cfDNA fragments that are prone to oxidation can easily penetrate the cancer cells and be expressed. The cfDNA should become a target for the antitumor therapy.

Changes of KEAP1/NRF2 and IKB/NF-κB Expression Levels Induced by Cell-Free DNA in Different Cell Types.

Cell-free DNA (cfDNA) is a circulating DNA of nuclear and mitochondrial origin mainly derived from dying cells. Recent studies have shown that cfDNA is a stress signaling DAMP (damage-associated molecular pattern) molecule. We report here that the expression profiles of cfDNA-induced factors NRF2 and NF-κB are distinct depending on the target cell's type and the GC-content and oxidation rate of the cfDNA. Stem cells (MSC) have shown higher expression of NRF2 without inflammation in response to cfDNA. In contrast, inflammatory response launched by NF-κB was dominant in differentiated cells HUVEC, MCF7, and fibroblasts, with a possibility of transition to massive apoptosis. In each cell type examined, the response for oxidized cfDNA was more acute with higher peak intensity and faster resolution than that for nonoxidized cfDNA. GC-rich nonoxidized cfDNA evoked a weaker and prolonged response with proinflammatory component (NF-κB) as predominant. The exploration of apoptosis rates after adding cfDNA showed that cfDNA with moderately increased GC-content and lightly oxidized DNA promoted cell survival in a hormetic manner. Novel potential therapeutic approaches are proposed, which depend on the current cfDNA content: either preconditioning with low doses of cfDNA before a planned adverse impact or eliminating (binding, etc.) cfDNA when its content has already become high.

An exposure to the oxidized DNA enhances both instability of genome and survival in cancer cells.

BACKGROUND: Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases and acts upon the cells. Response to cfDNA depends on concentrations and levels of the damage within cfDNA. Oxidized extracellular DNA acts as a stress signal and elicits an adaptive response. PRINCIPAL FINDINGS: Here we show that oxidized extracellular DNA stimulates the survival of MCF-7 tumor cells. Importantly, in cells exposed to oxidized DNA, the suppression of cell death is accompanied by an increase in the markers of genome instability. Short-term exposure to oxidized DNA results in both single- and double strand DNA breaks. Longer treatments evoke a compensatory response that leads to a decrease in the levels of chromatin fragmentations across cell populations. Exposure to oxidized DNA leads to a decrease in the activity of NRF2 and an increase in the activity of NF-kB and STAT3. A model that describes the role of oxidized DNA released from apoptotic cells in tumor biology is proposed. CONCLUSIONS/SIGNIFICANCE: Survival of cells with an unstable genome may substantially augment progression of malignancy. Further studies of the effects of extracellular DNA on malignant and normal cells are warranted.

Oxidized DNA induces an adaptive response in human fibroblasts.

Cell-free DNA (cfDNA) released from dying cells contains a substantial proportion of oxidized nucleotides, thus, forming cfDNA(OX). The levels of cfDNA(OX) are increased in the serum of patients with chronic diseases. Oxidation of DNA turns it into a stress signal. The samples of genomic DNA (gDNA) oxidized by Н2О2in vitro (gDNA(OX)) induce effects similar to that of DNA released from damaged cells. Here we describe the effects of gDNA(OX) on human fibroblasts cultivated in the stressful conditions of serum withdrawal. In these cells, gDNA(OX) evokes an adaptive response that leads to an increase in the rates of survival in serum starving cell populations as well as in populations irradiated at the dose of 1.2Gy. These effects are not seen in control populations of fibroblasts treated with non-modified gDNA. In particular, the exposure to gDNA(OX) leads to a decrease in the expression of the proliferation marker Ki-67 and an increase in levels of РСNА, a decrease in the proportion of subG1- and G2/M cells, a decrease in proportion of cells with double strand breaks (DSBs). Both gDNA(OX) and gDNA suppress the expression of DNA sensors TLR9 and AIM2 and up-regulate nuclear factor-erythroid 2 p45-related factor 2 (NRF2), while only gDNA(OX) inhibits NF-κB signaling. gDNA(OX) is a model for oxidized cfDNA(OX) that is released from the dying tumor cells and being carried to the distant organs. The systemic effects of oxidized DNA have to be taken into account when treating tumors. In particular, the damaged DNA released from irradiated cells may be responsible for an abscopal effects and a bystander mediated adaptive response seen in some cancer patients. These results indicate the necessity for the further study of the effects of oxidized DNA in both in vitro and in vivo systems.

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.

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.