Emotional Stress Induces Adaptive Response in Rat Lymphocytes to Subsequent Ionizing Radiation Exposure.

We studied the molecular mechanisms of cross-adaptation to ionizing radiation (1 Gy) of lymphocytes isolated from rats subjected to emotional stress. The effects of chronic (CES; various types of stress exposure) and acute (AES; forced swimming) emotional stress in rats on indicators of oxidative stress, cell death, and levels of NRF2 and NOX4 proteins involved in the development of the adaptive response were analyzed in isolated lymphocytes. It was found that stress induced an adaptive response in rat lymphocytes and triggered processes similar to the adaptive response induced by low doses of ionizing radiation: an increase in the level of oxidized DNA and cell death, as well as an increase in the content of NOX4 and NRF2 proteins. In animals subjected to emotional stress, suppressed DNA oxidation in response to irradiation, reduced levels of protective factor NRF2, as well as lymphocyte death were observed.

RSH ANTIOXIDANTS INCREASE INTRACELLULAR ROS LEVEL AND PROLIFERATION OF SP2/0 MYELOMA CELLS IN SERUM-FREE MEDIUM.

At present research we have demonstrated the link between introducing of low molecular weight RSH-antioxidants (N-acetylcysteine, glutathione) into serum-free medium composition, reactive oxygen species (ROS) generation and mouse myeloma SP2/0-SF cells proliferative activity. The presence of these compounds in the medium changed the pattern of ROS-activity in cells by concentration-dependent manner, and affected their proliferative characteristics. The optimal value of the proliferative activity was related to 0.2 mM for both thyols and not depended from the thyol-compound nature. Further increasing up from the found concentration optimum lead to growth inhibition with different expression for N-acetylcysteine and glutathione.

Properties of extracellular DNA from the cerebrospinal fluid and blood plasma during Parkinson's disease.

The cerebrospinal fluid of patients with Parkinson's disease was shown to contain extracellular DNA. Extracellular DNA concentration in the cerebrospinal fluid was 3.3-fold lower than in blood plasma from these patients. HPLC-mass spectrometry analysis showed that the pool of extracellular DNA from the liquor is characterized by a lower content of deoxythymidine, but greater amounts of deoxycytidine and deoxyguanosine than the pool of extracellular DNA from the plasma. The level of deoxyguanosine was 2 times lower than that of deoxycytidine (as differentiated from plasma extracellular DNA with similar content of these substances). Our findings indicate that extracellular DNA from the cerebrospinal fluid contains considerable amounts of modified deoxyguanosine. These data attest to significant differences in the quantitative and qualitative characteristics of extracellular DNA from the blood and cerebrospinal fluid of patients. Specific features of extracellular DNA from the cerebrospinal fluid of patients suggest its involvement in the pathogenesis of Parkinson's disease.

Oxidized extracellular DNA suppresses nitric oxide production by endothelial NO synthase (eNOS) in human endothelial cells (HUVEC).

Circulating DNA from patients with cardiovascular diseases reduce the synthesis of NO in endothelial cells, which is probably related to oxidative modification of DNA. To test this hypothesis, HUVEC cells were cultured in the presence of DNA containing ~1 (nonoxidized DNA), 700, or 2100 8-oxodG/10(6) nucleosides. Nonoxidized DNA stimulated the synthesis of NO, which was associated with an increase in the expression of endothelial NO synthase. Oxidized NO decreased the amount of mRNA and protein for endothelial NO synthase, but increased the relative content of its low active form. These changes were accompanied by reduction of NO production. These findings suggest that oxidative modification of circulating extracellular DNA contributes to endothelial dysfunction manifested in suppression of NO production.

[Circulating in blood plasma cell-free DNA in the pathogenesis of ischemic stroke: the role of the transcribed region of ribosomal repeat].

It has been established that DNA, in addition to its basic functions (storage and realization of genetic information), also carries CpG-rich sequences having immunopotentiating properties. In this study we investigated the dynamics of the quantitative and qualitative characteristics of cell-free DNA (cfDNA) circulating in blood plasma of patients with acute ischemic stroke compared with the biomechanics of their blood samples flow, cerebral infarct volume and dynamics of neurological disorders. The results obtained revealed a new drag-reducing function of the circulating cfDNA and its important role in a regulation of blood flow hydrodynamic resistance in conditions of disturbed cerebral circulation. Moreover, our results showed a dependence of cerebral infarct volume and clinical picture dynamics on the plasma concentration of transcribed region of ribosomal repeat CpG-rich sequences (rDNA). It was established a new function of rDNA fragments circulating in the total pool cfDNK, i.e., generation of the intercommunication between blood and brain cells to induce neuroprotection in ischemic stroke.

[Dimeric bisberzimidazoles: cytotoxicity and effects on DNA methylation in normal and cancer human cells].

Cancer cells are characterized by the hypermethylation of promoter regions of tumor suppressor genes. DNA methyltransferase inhibitors cause re-activation of these genes that allows considering DNA methyltransferases as targets for anticancer therapy. As it was previously shown by us, dimeric bisbenzimidazoles, DB(n), differing in length of the oligomethylene linker between the two bisbenzimidazole fragments (n--number of methylene groups in linker) effectively inhibit the methylation of DNA duplexes by murine methyltransferase Dnmt3a. Here, the cytotoxicity of some of these compounds, their penetration into cells and influence on the methylation of genomic DNA in fetal lung fibroblasts line F-977 and cervical cancer cells HeLa have been studied. In the 0-60 microM concentration range, only the DB(11) displayed a significant toxic effect on the normal cells, whereas the effect of DB(n) investigated on the cancer cells was not significant. Interestingly, the DB(1) and DB(3) to a small extent stimulate the proliferation of HeLa and F-977 cells, respectively. DB(1) and DB(3) display ability to penetrate into the nucleus of HeLa and F-977 cells and accumulate in various parts of the nuclei. DB(11) is not able to penetrate into the nuclei of these cells. The incubation of F-977 cells with 26 microM of DB(1) or DB(3) led to a decrease of the methylation of 18S rRNA gene, which is located in the region of DB(1) and DB(3) accumulation. A similar effect produces the same concentration of DB (3) in the F-977 cells. However, the overall level of genomic DNA methylation was not changed. These data suggest that DB(n) can be directed to act on specific genes demethylation and in the future may selectively inhibit the proliferation of cancer cells.

Emotional stress in rats changes concentration and composition of extracellular DNA circulating in blood plasma under normal conditions and in cerebral ischemia.

We studied quantitative and qualitative characteristics of extracellular DNA circulating in the blood plasma of Wistar rats under normal conditions, in psychoemotional stress (after 18 hours of aggressive conflict situation), and in acute cerebral ischemia. It was found that animals predisposed to psychoemotional stress normally have increased levels of antibodies against low excreted fragment of transcribed region of ribosomal DNA repeat rich in cytosine-guanine (CpG). A sharp increase in the level of circulating extracellular DNA was noted. Its increase was more pronounced during ischemia against the background of psychoemotional stress than in the control. These data suggest that multiple stress exposures experienced during the life can result in accumulation of GpG-rich sequences in the plasma of individuals predisposed to psychoemotional stress.

[DNA-signaling pathway mediating development of a radiation-induced bystander effect in human cells].

Low doses of ionizing radiation induce the adaptive effect (AE) development in human cells which is followed by a number of cell responses. These responses can be transmitted from irradiated cells to non-irradiated ones (bystander effect, BE). The major role in radiation-induced BE is played by an oxidative stress (OS) and a DNA-signaling pathway, in which extracellular DNA fragments (ecDNA) are the factors of stress-signalization. We propose the following sequence of events in this signaling system: irradiation-OS-DNA modification-apoptosis of irradiated cells-ecDNA-signal acceptance by non-irradiated cells-OS-DNA modification, etc. We observed a radiation-induced BE which is accompanied by DNA-signaling pathway in differentiated and undifferentiated human cells forming monolayer or suspension cultures. Here we discuss several aspects of the radiation-induced BE mechanism and its persistence possibilities.

[Overexpression of the nucleolar protein SURF-6 in mouse fibroblasts NIH/3T3 leads to stabilisation of intragenic transcribed spacers of the pre-rRNA].

SURF-6 is an evolutionary conserved nucleolar protein that is required for maintenance of cell viability, but its functional significance in mammals still remains illusive. In the present work we examined effects of SURF-6 overexpression in mouse NIH/3T3 fibroblasts transfected with two plasmids. The plasmid pUHrT62-1 encodes a tetracycline-dependant trans-activator, the protein rtTA, the plasmid pBI-SURF6--the genes of EGFP (enhanced green fluorescent protein) and of mouse SURF-6 which expression was controlled by the rtTA-responsive bi-directorial promoter. Western blot analysis showed that the SURF-6 level was severely augmented in cells transfected with pUHrT62-1 and pBI-SURF6 and incubated with the inducer--doxycycline opposed to the transfected but not-induced cells. The increase of SURF-6 was observed in 24 and 48 h after adding the inducer doxycycline. Dot-hybridization of isolated RNA with biotinilated oligonucleotide probes to various regions of mouse primarily pre-rRNA transcripts showed that overexpression of SURF-6 enhanced levels of the second intragenic transcribed spacer ITS2 in about seven folds and of the 5' external transcribed spacer 5'ETS in two folds. Amounts of fragments corresponding to 18S, 5.8S and 28S rRNA remained almost unchanged. These observations for the first time demonstrated that mammalian SURF-6 helps to stabilize or prevents premature cleavage of the pre-rRNA intragenic transcribed spacers, particularly of ITS2, similar to its homologue in S. cerevisiae the protein Rrp14. Today metazoan proteins that play a similar role in ribosome biogenesis, are not described.

[Bystander effect development in human mesenchymal stem cells after exposure to adaptive dose of X-radiation].

Transposition and mutual approaching of pericentromeric loci 1q12 of homological chromosomes from the nuclear membrane towards the nuclear centre as well as activation of the chromosomal nucleolus-forming regions (NFR) are observed in human mesenchymal stem cells (hMSCs) as an initial stages of the adaptive response (AR) after exposure to low doses of X-radiation (10 cGy). All these reactions are also induced after addition of cultivation medium from irradiated cells to intact bystander-cells and this phenomenon called bystander effect (BE). Recently the same AR and BE induction results were obtained for human G0-lymphocytes. All these data indicate the existence of universal reaction of homological chromosome loci transposition which was revealed during AR development in differentiated (lymphocytes) and non-differentiated (hMSCs) and also it shows possibility of radiational BE development in suspension and monolayer cell cultures upon addition of stress-signalization factors in incubation medium. We suppose that these factors are extracellular genome DNA fragments apoptotic cells.

[Ribosomal genes in the human genome: identification of four fractions, their organization in the nucleolus and metaphase chromosomes].

Completion of human genome reading stimulated intense studies in the field of functional genomics and characterization of individual genomes. Of considerable importance is the study of the complex of multicopy ribosomal genes (RGs), but its thorough analysis was not a task of the "Human Genome" program. In this short review we present our data on the copy number of rRNA genes in individual human genomes and on their heterogeneity in the functional respect. Fractions of active and potentially active RGs as well as fractions of inactive and silent RGs intensively methylated in the transcribed region are characterized. Their location in the nucleolus structures and in metaphase chromosomes is discussed.

Enhanced expression of iNOS in human endothelial cells during long-term culturing with extracellular DNA fragments.

NO synthesis by endothelial cells plays an important role in normal function of the cardiovascular system. This work was designed to evaluate the role of variations in properties of extracellular DNA in the regulation of NO synthesis. We studied the effect of four DNA samples with various base sequences (50 ng/ml) on functional activity of endothelial cells HUVEC during 24-h culturing. Human DNA fragments with high content of CG repeats increased intracellular content of NO and its metabolites (nitrites and nitrates) and accelerated oxidation of nitrites to nitrates. Changes in the content of NO metabolites after 24-h culturing was shown to depend on the expression of gene for inducible, but not for endothelial NO synthase. Increased expression of inducible NO synthase positively correlated with an increase in the content of mRNA for the adapter protein MyD88, but did not depend on TLR9 gene expression that encodes protein receptor for CG-DNA recognition. The intracellular concentration of MyD88 mRNA did not depend on the content of TLR9 mRNA. The existence of a variety of DNA-binding receptors apart from TLR9 receptor on the surface of endothelial cells was hypothesized. Activation of these receptors by extracellular DNA fragments stimulates expression of the adapter protein MyD88.

Extracellular DNA affects NO content in human endothelial cells.

Fragments of extracellular DNA are permanently released into the blood flow due to cell apoptosis and possible de novo DNA synthesis. To find out whether extracellular DNA can affect the synthesis of nitric oxide (NO), one of key vascular tone regulators, we studied in vitro effects of three artificial DNA probes with different sequences and 10 samples of extracellular DNA (obtained from healthy people and patients with hypertension and atherosclerosis) on NO synthesis in endothelial cell culture (HUVEC). For detection of NO in live cells and culture medium, we used a NO-specific agent CuFL penetrating into the cells and forming a fluorescent product FL-NO upon interaction with NO. Human genome DNA fragments affected the content of NO in endothelial cells; this effect depended on both the base sequence and concentration of DNA fragments. Addition of artificial DNA and extracellular DNA from healthy people into the cell culture in a low concentration (5 ng/ml) increased the detected NO concentration by 4-fold at most. Cytosine-guanine (CG)-rich fragment of the transcribed sequence of ribosomal repeat was the most powerful NO-inductor. The effect of DNA fragments on NO synthesis was comparable with that of low doses of oxidizing agents, H(2)O(2) and 17ß-estradiol. Extracellular DNA samples obtained from patients with hypertension and atherosclerosis decreased NO content in cells and medium by 1.3-28 times compared to the control; the effect correlated with the content of CG-rich sequences.

Copy number variations of satellite III (1q12) and ribosomal repeats in health and schizophrenia.

OBJECTIVE: Earlier we studied the copy number variations (CNVs) of ribosomal repeat (rDNA) and the satellite III fragment (1q12) (f-SatIII) in the cells of schizophrenia patients (SZ) and healthy controls (HC). In the present study we pursued two main objectives: (1) to confirm the increased rDNA and decreased f-SatIII content in the genomes of enlarged SZ and HC samples and (2) to compare the rDNA and f-SatIII content in the same DNA samples of SZ and HC individuals. METHODS: We determined the rDNA CN and f-SatIII content in the genomes of leukocytes of 1770 subjects [HC (N = 814) and SZ (N = 956)]. Non-radioactive quantitative hybridization method (NQH) was applied for analysis of the various combinations of the two repeats sizes in SZ and HC groups. RESULTS: f-SatIII in human leukocytes (N = 1556) varies between 5.7 and 44.7 pg/ng DNA. RDNA CN varies between 200 and 896 (N = 1770). SZ group significantly differ from the HC group by lower f-SatIII content and by rDNA abundance. The f-SatIII and rDNA CN are not randomly combined in the genome. Higher rDNA CN values are associated with higher f-SatIII index values in SZ and HC. The f-SatIII variation interval in SZ group increases significantly in the subgroup with the high rDNA CN index values (>300 copies). CONCLUSION: Schizophrenia patients' genomes contain low number of f-SatIII copies corresponding with a large ribosomal repeats CN. A scheme is proposed to explain the low f-SatIII content in SZ group against the background of high rDNA CN.

[CpG-DNA inhibits cell reactions accompanied with the development of the adaptive response in human lymphocytes after low-dose X-ray exposure].

The lymphocytes of peripheral blood of healthy donors were influenced by X-ray radiation (10 cGy) or a fragments of the transcribed region of rDNA (TRrDNA) transmitted to the incubation medium of non-irradiated cells. Both factors induced transposition of the loci 1q12 of homologous chromosomes from the membrane to the centre of the nucleus in lymphocytes; produced the activation of the genes TLR9 and MyD88 expression, the chromosomal nucleolus-forming regions, TNF-alpha and caspase-3; and also increased nuclease activity and synthesis RNA of the cells. However all the investigated reaction in the cells did not developed during the synergetic radiation and TRrDNA but the activity level of the cytokine TNF-alpha was increasing. The reactions of human lymphocytes on the induced influence are discussed herein.

[The response of human cancer stem cells on low-dose X-ray exposure].

Recently we found that transposition of homologous chromosomes 1q12 loci towards the nuclear centre and activation of the chromosomal nucleolus-forming regions (NFR) are observed in human lymphocytes after exposure to low doses of X-radiation (10 cGy). These cell reactions were studied for human breast cancer stem cell cultures. There are two cell types in cell culture from single donor: with two (type 1) and three (type 2) loci of 1q12. It was shown that an adaptive response induced by X-ray irradiation is developed only in cells of the type 1 but not in type 2 ones after 3 and 10 cGy doses. We observed a considerable death of cell type 2 after low-dose exposure. Activation of the NFR in breast cancer stem cells after irradiation was not found. In this paper we discuss features of studied cancer stem cells lines and their responses to low doses of ionizing radiation.

[Haemodynamic role of blood-plasma circulating cell-free DNA and contained therein high-molecular-weight CpG-rich fraction in pathogenesis of arterial hypertension and atherosclerosis obliterans of carotid arteries].

The hydrodynamic resistance (HR) of blood is one of the components of the total peripheral resistance. High-molecular-weight DNA appears to decrease the HR in accordance with the Toms's effect. The present study was undertaken to investigate the HR and properties of cell-free DNA circulating in the blood plasma (hereinafter referred to as pDNA) of the control donors, patients suffering from either arterial hypertension (AH) alone or that combined with atherosclerotic lesions of the carotid arteries (CAs). Within the normal concentrations of pDNA, we revealed an inverse dependence of the HR thereupon and upon the content in pDNA of the high-molecular-weight CpG-rich fraction (CpG-DNA), i. e., a transcribed region of the ribosomal repeat (rDNA). A decrease or an increase in the pDNA concentration in all the patients examined was accompanied by an elevation of the rDNA concentration in the blood plasma. Exceeding a certain level thereof appeared to give rise to an increase in both the HR and arterial pressure (AP). Patients presenting with degree I essential AH were found to have a decreased endonuclease activity of the blood plasma, with the pDNA concentration being more than two-fold higher with no change in the rDNA content. Their HR appeared to be increased (p<0.01). Patients diagnosed as having degree II AH were characterized by a normal or decreased level of pDNA and an elevated content of pDNA, with the HR being slightly lowered. In patients presenting with atherosclerosis obliterans of the ACs, the initial manifestations of the lesions of the carotid arteries were typically revealed on the background of a lowered HR (p<0.05). All patients suffering from atherosclerotic lesions of the ACs could be subdivided into two groups, which in our opinion is probably associated with different various mechanisms of destructive damage to the arterial intima. In some of them, the pDNA concentration does not differ from the normal values, but in its composition, there is an increased content of rDNA, elevating as obliteration of the vessels' lumen increases, with the HR being decreased. The majority of them have degree II AH. In others, the pDNA concentration is by an order of magnitude higher than the normal values, while the rDNA content in pDNA is decreased, with the HR being elevated. Most of them have degree III AH. Pronounced and rough stenoses take an asymptomatic course in patients with decreased values of the HR and a slightly elevated level of pDNA and/or rDNA in the blood plasma. A higher level thereof leads to a rise in the HR and to the appearance of neurological symptomatology. Hence, CpG-DNA circulating in the composition of pDNA is a constantly acting endogenous blood factor decreasing the HR (the Toms's effect) and normalizing AP under physiological conditions, being however a cause of their increase and impairment of blood circulation in the pathogenesis of AH and atherosclerosis obliterans of the CAs.

Accumulation of Circulating Cell-Free CpG-Enriched Ribosomal DNA Fragments on the Background of High Endonuclease Activity of Blood Plasma in Schizophrenic Patients.

INTRODUCTION: Schizophrenia (SZ) increases the level of cell death, leading to an increase in the concentration of circulating cell-free DNA (cfDNA). Ribosomal DNA (rDNA) contains many unmethylated CpG motifs that stimulate TLR9-MyD88-NF-κB signaling and the synthesis of proinflammatory cytokines. The number of rDNA copies in the genomes of SZ patients is increased; therefore, we expect that the concentration of cell-free rDNA in the plasma of the SZ patients also increases. This may be one of the explanations of the proinflammatory cytokine increase that is often observed in SZ. The major research question is what is the rDNA copy number in cfDNA (cf-rDNA CN) and its putative role in schizophrenia? Materials and Methods. We determined cfDNA concentration (RNase A/proteinase K/solvent extraction; fluorescent dye PicoGreen) and endonuclease activity (NA) of blood plasma (radial diffusion method) in the untreated male SZ group (N = 100) and in the male healthy control group (HC) (N = 96). Blood leukocyte DNA and cfDNA rDNA CN were determined with nonradioactive quantitative hybridization techniques. Plasma concentration of cf-rDNA was calculated. RESULTS: In the subjects from the SZ group, the mean cfDNA plasma concentration was twofold higher and NA of the plasma was fourfold higher than those in the healthy controls. rDNA CN in the blood leukocyte genome and in the cfDNA samples in the SZ group was significantly higher than that in the HC group. cf-rDNA concentration was threefold higher in the SZ group. CONCLUSION: Despite the abnormally high endonuclease activity in the blood plasma of SZ patients, the circulating cfDNA concentration is increased. Fragments of cf-rDNA accumulate in the blood plasma of SZ patients. Potentially, SZ patients' cfDNA should be a strong stimulating factor for the TLR9-MyD88-NF-κB signaling pathway.

Oxidized Cell-Free DNA Role in the Antioxidant Defense Mechanisms under Stress.

The present study focuses on the investigation of the oxidized cell-free DNA (cfDNA) properties in several experimental models, including cultured cerebellum cells, peripheral blood lymphocytes (PBL), plasma, and hippocampus under an acute and chronic unpredictable stress model in rats. Firstly, our study shows that Spectrum Green fluorescence-labeled oxidized cfDNA fragments were transferred into the cytoplasm of 80% of the cerebellum culture cells; meanwhile, the nonoxidized cfDNA fragments do not pass into the cells. Oxidized cfDNA stimulates the antioxidant mechanisms and induction of transcription factor NRF2 expression, followed by an activation of NRF2 signaling pathway genes-rise of Nrf2 and Hmox1 gene expression and consequently NRF2 protein synthesis. Secondly, we showed that stress increases plasma cfDNA concentration in rats corresponding with the duration of the stress exposure. At the same time, our study did not reveal any significant changes of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) level in PBL of rats under acute or chronic stress, probably due to the significantly increased Nrf2 expression, that we found in such conditions. 8-oxodG is one of the most reliable markers of DNA oxidation. We also found an increased level of 8-oxodG in the hippocampal homogenates and hippocampal dentate gyrus in rats subjected to acute and chronic stress. Taken together, our data shows that oxidized cfDNA may play a significant role in systemic and neuronal physiological mechanisms of stress and adaptation.

[Extracellular DNA fragments from culture medium of low-dose irradiated human lymphocyte trigger instigating of the oxidative stress and the adaptive response in non-irradiated bystander lymphocytes].

We have previously shown that the induced by X-ray radiation (10 cGy) in human lymphocytes reactions of transposition of the loci of homologous chromosomes from the membrane to the centre of the nucleus, and activation of the chromosomal nucleolus-forming regions (NFR) are transmitted via DNA fragments to the nonirradiated cells--the so-called bystander effect (BE). In the present study, the blockade of the oxidative stress (OS) with alpha-tocopherol prior to irradiation or treatment with H2O2 induced no effects of either chromosomal loci transposition or activation of the NFR; neither in the presence of alpha-tocopherol were these reactions induced by the addition of the DNA fragments from the growth medium of the exposed (X-irradiated or H2O2-treated) lymphocytes to the bystander cells. Moreover, after inhibiting the activity of caspase 3 in the H2O2-treated/irradiated lymphocytes or suppression of the toll-like receptors (TLR9) in their bystander cells, we observed no transposition of the chromosomal loci. Based on the reported and previously obtained findings we suggest that the induced OS specifically modifies nuclear DNA, instigating the mechanisms of the adaptive response (AR) and apoptosis of the radiation-sensitive lymphocytes, while the interaction of the DNA fragments released therefrom with the TLR9 of the bystander cells leads to the development of the OS in last, to be followed by the AR (BE). Possibilities of such a pathway are discussed herein.