Antipsychotics Affect Satellite III (1q12) Copy Number Variations in the Cultured Human Skin Fibroblasts.

The fragment of satellite III (f-SatIII) is located in pericentromeric heterochromatin of chromosome 1. Cell with an enlarged f-SatIII block does not respond to various stimuli and are highly stress-susceptible. The fraction of f-SatIII in the cells of schizophrenia patients changed during antipsychotic therapy. Therefore, antipsychotics might reduce the f-SatIII content in the cells. We studied the action of haloperidol, risperidone and olanzapine (3 h, 24 h, 96 h) on human skin fibroblast lines (n = 10). The f-SatIII contents in DNA were measured using nonradioactive quantitative hybridization. RNASATIII were quantified using RT-qPCR. The levels of DNA damage markers (8-oxodG, γ-H2AX) and proteins that regulate apoptosis and autophagy were determined by flow cytometry. The antipsychotics reduced the f-SatIII content in DNA and RNASATIII content in RNA from HSFs. After an exposure to the antipsychotics, the autophagy marker LC3 significantly increased, while the apoptosis markers decreased. The f-SatIII content in DNA positively correlated with RNASATIII content in RNA and with DNA oxidation marker 8-oxodG, while negatively correlated with LC3 content. The antipsychotics arrest the process of f-SatIII repeat augmentation in cultured skin fibroblasts via the transcription suppression and/or through upregulated elimination of cells with enlarged f-SatIII blocks with the help of autophagy.

Association of NEF2L2 Rs35652124 Polymorphism with Nrf2 Induction and Genotoxic Stress Biomarkers in Autism.

Increased oxidative/genotoxic stress is known to impact the pathophysiology of ASD (autism spectrum disorder). Clinical studies, however, reported limited, heterogeneous but promising responses to treatment with antioxidant remedies. We determined whether the functional polymorphism of the Nrf2 gene, master regulator of anti-oxidant adaptive reactions to genotoxic stress, links to the genotoxic stress responses and to an in vitro effect of a NRF2 inductor in ASD children. Oxidative stress biomarkers, adaptive responses to genotoxic/oxidative stress, levels of master antioxidant regulator Nrf2 and its active form pNrf2 before and after inducing by dimethyl fumarate (DMF), and promotor rs35652124 polymorphism of NFE2L2 gene encoding Nrf2 were studied in children with ASD (n = 179). Controls included healthy adults (n = 101). Adaptive responses to genotoxicity as indicated by H2AX and cytoprotection by NRF2 contents positively correlated in ASD children with a Spearman coefficient of R = 0.479 in T+, but not CC genotypes. ASD children with NRF2 rs35652124 CC genotype demonstrated significantly higher H2AX content (0.652 vs. 0.499 in T+) and pNrf2 induction by DMF, lowered 8-oxo-dG concentration in plasma and higher cfDNA/plasma nuclease activity ratio. Our pilot findings suggest that in ASD children the NEF2L2 rs35652124 polymorphism impacts adaptive responses that may potentially link to ASD severity. Our data warrant further studies to reveal the potential for NEF2L2 genotype-specific and age-dependent repurposing of DMF and/or other NRF2-inducing drugs.

Effects of Aqueous Dispersions of C60, C70, and Gd@C82 Fullerenes on DNA Oxidative Damage/Repair and Apoptosis in Human Embryonic Lung Fibroblasts.

Fullerenes and metallofullerenes play an active role in homeostasis of reactive oxygen species and may cause oxidative damage to cells. As pristine fullerenes are a basis for derivatization, studying oxidative DNA damage/repair and apoptosis is important in terms of genotoxicity and cytotoxicity for their biomedical application. Aqueous dispersions of C60, C70, and Gd@C82 (5 nM and 1.5 µM) were cultured with human fetal lung fibroblasts for 1, 3, 24, and 72 h. Oxidative DNA damage/repair was assessed through concentration of 8-oxodG, double-strand breaks, and activation of BRCA1. Activity of apoptosis was assessed through the BCL2/BAX ratio. All three fullerenes caused oxidative modification of DNA at the early stages; C60 caused the most long-term damage, Gd@C82 caused the most short-term damage, and C70 caused "wave-like" dynamics. The dynamics of DNA repair correlated with the dynamics of oxidative damage, but Gd@C82 caused more prolonged activation of the repair system than C60 or C70. The oxidative toxicity of Gd@C82, is minor and the oxidative toxicity of C60 is mild and short-term, in contrast to C70. In relation to the studied effects, the fullerenes can be arranged in a safety row of Gd@C82 > C60 > C70.

In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls-Part 2: Adaptive Response.

Oxidized in vitro genomic DNA (gDNA) is known to launch an adaptive response in human cell cultures. The cfDNA extracted from the plasma of schizophrenic patients (sz-cfDNA) and healthy controls (hc-cfDNA) contains increased amounts of 8-oxodG, a DNA-oxidation marker. The aim of the research was answering a question: can the human cfDNA isolated from blood plasma stimulate the adaptive response in human cells? In vitro responses of ten human skin fibroblasts (HSFs) and four peripheral blood mononuclear cell (PBMC) lines after 1-24 h of incubation with sz-cfDNA, gDNA and hc-cfDNA containing different amounts of 8-oxodG were examined. Expressions of RNA of eight genes (NOX4, NFE2L2, SOD1, HIF1A, BRCA1, BRCA2, BAX and BCL2), six proteins (NOX4, NRF2, SOD1, HIF1A, γH2AX and BRCA1) and DNA-oxidation marker 8-oxodG were analyzed by RT-qPCR and flow cytometry (when analyzing the data, a subpopulation of lymphocytes (PBL) was identified). Adding hc-cfDNA or sz-cfDNA to HSFs or PBMC media in equal amounts (50 ng/mL, 1-3 h) stimulated transient synthesis of free radicals (ROS), which correlated with an increase in the expressions of NOX4 and SOD1 genes and with an increase in the levels of the markers of DNA damage γH2AX and 8-oxodG. ROS and DNA damage induced an antioxidant response (expression of NFE2L2 and HIF1A), DNA damage response (BRCA1 and BRCA2 gene expression) and anti-apoptotic response (changes in BAX and BCL2 genes expression). Heterogeneity of cells of the same HSFs or PBL population was found with respect to the type of response to (sz,hc)-cfDNA. Most cells responded to oxidative stress with an increase in the amount of NRF2 and BRCA1 proteins along with a moderate increase in the amount of NOX4 protein and a low amount of 8-oxodG oxidation marker. However, upon the exposure to (sz,hc)-cfDNA, the size of the subpopulation with apoptosis signs (high DNA damage degree, high NOX4 and low NRF2 and BRCA1 levels) also increased. No significant difference between the responses to sz-cfDNA and hc-cfDNA was observed. Sz-cfDNA and hc-cfDNA showed similarly high bioactivity towards fibroblasts and lymphocytes. Conclusion: In cultured human cells, hc-cfDNA and sz-cfDNA equally stimulated an adaptive response aimed at launching the antioxidant, repair, and anti-apoptotic processes. The mediator of the development of the adaptive response are ROS produced by, among others, NOX4 and SOD1 enzymes.

Ribosomal DNA Abundance in the Patient's Genome as a Feasible Marker in Differential Diagnostics of Autism and Childhood-Onset Schizophrenia.

Introduction: Differential diagnostics of early-onset schizophrenia and autism spectrum disorders (ASD) are a problem of child psychiatry. The prognosis and relevant treatment are to a large degree determined by the correctness of diagnosis. We found earlier that leucocyte DNA of adult schizophrenia patients contained significantly larger copy numbers of ribosomal repeats (rDNA) coding for rRNA, than DNA of mentally healthy controls. Aim: To compare the contents of ribosomal repeats in the leucocyte DNA of children with schizophrenia, children with ASD, and healthy age-matched controls to estimate the possibility of using this genetic trait in the differential diagnostics of the two types of disorders. Patients and methods: Blood samples of patients with infantile autism (A­F84.0 according to ICD-10, N = 75) and with childhood-onset schizophrenia (SZ­F20.8 according to ICD-10, N = 43) were obtained from the Child Psychiatry Department of the Mental Health Research Center. The healthy control blood samples (HC, N = 86) were taken from the Research Centre for Medical Genetics collection. The recruitment of cases was based on the clinical psychopathologic approach. DNA was extracted from blood leukocytes with organic solvents. Nonradioactive quantitative hybridization technique was applied for determining the abundance of ribosomal repeats in the genomes. Statistical processing was performed using StatPlus, Statgraphics and MedCalc. Findings: DNA derived from SZ cases contained 565 ± 163 rDNA copies, which is significantly (p < 10−6) higher than the rDNA content in ASD cases (405 ± 109 copies) and controls (403 ± 86 copies). The HC and A groups did not differ by rDNA copy number (p > 0.4). The genetic trait "rDNA copy number in patient's genome" can potentially be applied as an additional marker in differential diagnostics of childhood-onset schizophrenia and autism spectrum disorders.

Mild cognitive impairment is associated with low copy number of ribosomal genes in the genomes of elderly people.

Introduction: Mild cognitive impairments (MCI) accompanying aging are associated with oxidative stress. The ability of cells to respond to stress is determined by the protein synthesis level, which depends on the ribosomes number. Ribosomal deficit was documented in MCI. The number of ribosomes depends, together with other factors, on the number of ribosomal genes copies. We hypothesized that MCI is associated with low rDNA CN in the elderly person genome. Materials and Methods: rDNA CN and the telomere repeat (TR) content were determined in the DNA of peripheral blood leukocytes of 93 elderly people (61-91 years old) with MCI and 365 healthy volunteers (16-91 years old). The method of non-radioactive quantitative hybridization of DNA with biotinylated DNA probes was used for the analysis. Results: In the MCI group, rDNA CN (mean 329 ± 60; median 314 copies, n = 93) was significantly reduced (p < 10-15) compared to controls of the same age with preserved cognitive functions (mean 412 ± 79; median 401 copies, n = 168) and younger (16-60 years) control group (mean 426 ± 109; median 416 copies, n = 197). MCI is also associated with a decrease in TR DNA content. There is no correlation between the content of rDNA and TR in DNA, however, in the group of DNA samples with rDNA CN > 540, TR content range was significantly narrowed compared to the rest of the sample. Conclusion: Mild cognitive impairment is associated with low ribosomal genes copies in the elderly people genomes. A low level of rDNA CN may be one of the causes of ribosomal deficit that was documented in MCI. The potential possibilities of using the rDNA CN indicator as a prognostic marker characterizing human life expectancy are discussed.

NADPH-oxidase 4 gene over-expression in peripheral blood lymphocytes of the schizophrenia patients.

INTRODUCTION: Increased systemic oxidative stress is common in schizophrenia (SZ) patients. NADPH-oxidase 4 (NOX4) is the cell oxidoreductase, catalyzing the hydrogen peroxide formation. Presumably, NOX4 is the main oxidative stress factor in a number of diseases such as cardiovascular diseases and cancer. We hypothesized that NOX4 may be involved in the oxidative stress development caused by the disease in the schizophrenic patients' peripheral blood lymphocytes (PBL). MATERIALS AND METHODS: The SZ group included 100 patients (68 men and 32 women aged 28 ± 11 years). The control group included 60 volunteers (35 men and 25 women aged 25 ± 12 years). Flow cytometry analysis (FCA) was used for DNA damage markers (8-oxodG, É£H2AX), pro- and antiapoptotic proteins (BAX1 and BCL2) and the master-regulator of anti-oxidant response NRF2 detection in the lymphocytes of the untreated SZ patients (N = 100) and the healthy control (HC, N = 60). FCA and RT-qPCR were used for NOX4 and RNANOX4 detection in the lymphocytes. RT-qPCR was used for mtDNA quantitation in peripheral blood mononuclear cells. Cell-free DNA concentration was determined in blood plasma fluorimetrically. RESULTS: 8-oxodG, NOX4, and BCL2 levels in the PBL in the SZ group were higher than those in the HC group (p < 0.001). É£H2AX protein level was increased in the subgroup with high 8-oxodG (p<0.02) levels and decreased in the subgroup with low 8-oxodG (p <0.0001) levels. A positive correlation was found between 8-oxodG, É£H2AX and BAX1 levels in the SZ group (p <10-6). NOX4 level in lymphocytes did not depend on the DNA damage markers values and BAX1 and BCL2 proteins levels. In 15% of PBL of the HC group a small cellular subfraction was found (5-12% of the total lymphocyte pool) with high DNA damage level and elevated BAX1 protein level. The number of such cells was maximal in PBL samples with low NOX4 protein levels. CONCLUSION: Significant NOX4 gene expression was found a in SZ patients' lymphocytes, but the corresponding protein is probably not a cause of the DNA damage.

In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls.

Schizophrenia is associated with low-grade systemic inflammation. Circulating cell-free DNA (c-cfDNA) belongs to the DAMP class. The major research question was: can the c-cfDNA of schizophrenic patients (sz-cfDNA) stimulate the DNA sensor genes, which control the innate immunity? We investigated the in vitro response of ten human skin fibroblast (HSF) lines to five DNA probes containing different amounts of a GC-rich marker (the ribosomal repeat) and a DNA oxidation marker (8-oxodG) including sz-cfDNA and healthy control c-cfDNA (hc-cfDNA) probes. After 1 h, 3 h, and 24 h of incubation, the expression of 6 protein genes responsible for cfDNA transport into the cell (EEA1 and HMGB1) and the recognition of cytosolic DNA (TLR9, AIM2, STING and RIG-I) was analyzed at the transcriptional (RT-qPCR) and protein level (flow cytometry and fluorescence microscopy). Additionally, we analyzed changes in the RNA amount of 32 genes (RT-qPCR), which had been previously associated with different cellular responses to cell-free DNA with different characteristics. Adding sz-cfDNA and hc-cfDNA to the HSF medium in equal amounts (50 ng/mL) blocked endocytosis and stimulated TLR9 and STING gene expression while blocking RIG-I and AIM2 expression. Sz-cfDNA and hc-cfDNA, compared to gDNA, demonstrated much stronger stimulated transcription of genes that control cell proliferation, cytokine synthesis, apoptosis, autophagy, and mitochondrial biogenesis. No significant difference was observed in the response of the cells to sz-cfDNA and hc-cfDNA. Sz-cfDNA and hc-cfDNA showed similarly high biological activity towards HSFs, stimulating the gene activity of TLR9 and STING DNA sensor proteins and blocking the activity of the AIM2 protein gene. Since the sz-cfDNA content in the patients' blood is several times higher than the hc-cfDNA content, sz-cfDNA may upregulate pro-inflammatory cytokines in schizophrenia.

The Psychoemotional Stress-Induced Changes in the Abundance of SatIII (1q12) and Telomere Repeats, but Not Ribosomal DNA, in Human Leukocytes.

INTRODUCTION: As shown earlier, copy number variations (CNV) in the human satellite III (1q12) fragment (f-SatIII) and the telomere repeat (TR) reflects the cell's response to oxidative stress. The contents of f-SatIII and TR in schizophrenic (SZ) patients were found to be lower than in healthy controls (HC) in previous studies. The major question of this study was: 'What are the f-SatIII and TR CNV dynamic changes in human leukocytes, depending on psychoemotional stress?' MATERIALS AND METHODS: We chose a model of psychoemotional stress experienced by second-year medical students during their exams. Blood samples were taken in stressful conditions (exams) and in a control non-stressful period. Biotinylated probes were used for f-SatIII, rDNA, and TR quantitation in leukocyte DNA by non-radioactive quantitative hybridization in SZ patients (n = 97), HC (n = 97), and medical students (n = 17, n = 42). A flow cytometry analysis was used for the oxidative stress marker (NOX4, 8-oxodG, and γH2AX) detection in the lymphocytes of the three groups. RESULTS: Oxidative stress markers increased significantly in the students' lymphocytes during psychoemotional stress. The TR and f-SatIII, but not the rDNA, contents significantly changed in the DNA isolated from human blood leukocytes. After a restoration period (post-examinational vacations), the f-SatIII content decreased, and the TR content increased. Changes in the blood cells of students during examinational stress were similar to those in SZ patients during an exacerbation of the disease. CONCLUSIONS: Psychoemotional stress in students during exams triggers a universal mechanism of oxidative stress. The oxidative stress causes significant changes in the f-SatIII and TR contents, while the ribosomal repeat content remains stable. A hypothesis is proposed to explain the quantitative polymorphisms of f-SatIII and TR contents under transient (e.g., students' exams) or chronic (in SZ patients) stress. The changes in the f-SatIII and TR copy numbers are non-specific events, irrespective of the source of stress. Thus, our findings suggest that the psychoemotional stress, common in SZ patients and healthy students during exams, but not in a schizophrenia-specific event, was responsible for the changes in the repeat contents that we observed earlier in SZ patients.

The Role of Human Satellite III (1q12) Copy Number Variation in the Adaptive Response during Aging, Stress, and Pathology: A Pendulum Model.

The pericentric satellite III (SatIII or Sat3) and II tandem repeats recently appeared to be transcribed under stress conditions, and the transcripts were shown to play an essential role in the universal stress response. In this paper, we review the role of human-specific SatIII copy number variation (CNV) in normal stress response, aging and pathology, with a focus on 1q12 loci. We postulate a close link between transcription of SatII/III repeats and their CNV. The accrued body of data suggests a hypothetical universal mechanism, which provides for SatIII copy gain during the stress response, alongside with another, more hypothetical reverse mechanism that might reduce the mean SatIII copy number, likely via the selection of cells with excessively large 1q12 loci. Both mechanisms, working alternatively like swings of the pendulum, may ensure the balance of SatIII copy numbers and optimum stress resistance. This model is verified on the most recent data on SatIII CNV in pathology and therapy, aging, senescence and response to genotoxic stress in vitro.

The Phosphonate Derivative of C60 Fullerene Induces Differentiation towards the Myogenic Lineage in Human Adipose-Derived Mesenchymal Stem Cells.

Inductors of myogenic stem cell differentiation attract attention, as they can be used to treat myodystrophies and post-traumatic injuries. Functionalization of fullerenes makes it possible to obtain water-soluble derivatives with targeted biochemical activity. This study examined the effects of the phosphonate C60 fullerene derivatives on the expression of myogenic transcription factors and myogenic differentiation of human mesenchymal stem cells (MSCs). Uptake of the phosphonate C60 fullerene derivatives in human MSCs, intracellular ROS visualization, superoxide scavenging potential, and the expression of myogenic, adipogenic, and osteogenic differentiation genes were studied. The prolonged MSC incubation (within 7-14 days) with the C60 pentaphoshonate potassium salt promoted their differentiation towards the myogenic lineage. The transcription factors and gene expressions determining myogenic differentiation (MYOD1, MYOG, MYF5, and MRF4) increased, while the expression of osteogenic differentiation factors (BMP2, BMP4, RUNX2, SPP1, and OCN) and adipogenic differentiation factors (CEBPB, LPL, and AP2 (FABP4)) was reduced or did not change. The stimulation of autophagy may be one of the factors contributing to the increased expression of myogenic differentiation genes in MSCs. Autophagy may be caused by intracellular alkalosis and/or short-term intracellular oxidative stress.

Effects of Aqueous Dispersions of C60, C70 and Gd@C82 Fullerenes on Genes Involved in Oxidative Stress and Anti-Inflammatory Pathways.

BACKGROUND: Fullerenes and metallofullerenes can be considered promising nanopharmaceuticals themselves and as a basis for chemical modification. As reactive oxygen species homeostasis plays a vital role in cells, the study of their effect on genes involved in oxidative stress and anti-inflammatory responses are of particular importance. METHODS: Human fetal lung fibroblasts were incubated with aqueous dispersions of C60, C70, and Gd@C82 in concentrations of 5 nM and 1.5 µM for 1, 3, 24, and 72 h. Cell viability, intracellular ROS, NOX4, NFκB, PRAR-γ, NRF2, heme oxygenase 1, and NAD(P)H quinone dehydrogenase 1 expression have been studied. RESULTS & CONCLUSION: The aqueous dispersions of C60, C70, and Gd@C82 fullerenes are active participants in reactive oxygen species (ROS) homeostasis. Low and high concentrations of aqueous fullerene dispersions (AFD) have similar effects. C70 was the most inert substance, C60 was the most active substance. All AFDs have both "prooxidant" and "antioxidant" effects but with a different balance. Gd@C82 was a substance with more pronounced antioxidant and anti-inflammatory properties, while C70 had more pronounced "prooxidant" properties.

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.

Oxidized cell-free DNA as a stress-signaling factor activating the chronic inflammatory process in patients with autism spectrum disorders.

BACKGROUND: Autism spectrum disorders (ASD) are known to be associated with an inflammatory process related to immune system dysfunction. This study's aim was to investigate the role of cell-free DNA in chronic inflammatory process in ASD patients. METHODS: The study included 133 ASD patients and 27 healthy controls. Sixty-two ASD patients were demonstrated to have mild-to-moderate disease severity (group I) and 71 individuals to have severe ASD (group II). Plasma cell-free (cf) DNA characteristics, plasma cytokine concentrations, expression of the genes for NFкB1 transcription factor and pro-inflammatory cytokines TNFα, IL-1ß and IL-8 in peripheral blood lymphocytes (PBL) of ASD patients, and unaffected controls were investigated. Additionally, in vitro experiments with oxidized DNA supplementation to PBL cultures derived from ASD patients and healthy controls were performed. RESULTS: The data indicates that ASD patients have demonstrated increased cfDNA concentration in their circulation. cfDNA of patients with severe ASD has been characterized by a high abundance of oxidative modification. Furthermore, ASD patients of both groups have shown elevated plasma cytokine (IL-1ß, IL-8, IL-17A) levels and heightened expression of genes for NFкB1 nuclear factor and pro-inflammatory cytokines TNFα, IL-1ß, and IL-8 in PBL. In vitro experiments have shown that NF-κB/cytokine mRNA expression profiles of ASD patient PBL treated with oxidized DNA fragments were significantly different from those of healthy controls. CONCLUSIONS: It may be proposed that oxidized cfDNA plays a role of stress-signaling factor activating the chronic inflammatory process in patients with ASD.

Effects of Functionalized Fullerenes on ROS Homeostasis Determine Their Cytoprotective or Cytotoxic Properties.

BACKGROUND: Functionalized fullerenes (FF) can be considered regulators of intracellular reactive oxygen species (ROS) homeostasis; their direct oxidative damage-as well as regulation of oxidant enzymes and signaling pathways-should be considered. METHODS: Uptake of two water-soluble functionalized C70 fullerenes with different types of aromatic addends (ethylphenylmalonate and thienylacetate) in human fetal lung fibroblasts, intracellular ROS visualization, superoxide scavenging potential, NOX4 expression, NRF2 expression, oxidative DNA damage, repair genes, cell proliferation and cell cycle were studied. RESULTS & CONCLUSION: The intracellular effects of ethylphenylmalonate C70 derivative (FF1) can be explained in terms of upregulated NOX4 activity. The intracellular effects of thienylacetate C70 derivative (FF2) can be probably resulted from its superoxide scavenging potential and inhibition of lipid peroxidation. FF1 can be considered a NOX4 upregulator and potential cytotoxicant and FF2, as a superoxide scavenger and a potential cytoprotector.

Copy Number Variation of Satellite III (1q12) in Patients With Schizophrenia.

Introduction: It was shown that copy number variations (CNVs) of human satellite III (1q12) fragment (f-SatIII) reflects the human cells response to stress of different nature and intensity. Patients with schizophrenia (SZ) experience chronic stress. The major research question: What is the f-SatIII CNVs in human leukocyte as a function of SZ? Materials and Methods: Biotinylated pUC1.77 probe was used for f-SatIII quantitation in leukocyte DNA by the non-radioactive quantitative hybridization for SZ patients (N = 840) and healthy control (HC, N = 401). SZ-sample included four groups. Two groups: first-episode drug-naïve patients [SZ (M-)] and medicated patients [SZ (M+)]. The medical history of these patients did not contain reliable confirmed information about fetal hypoxia and obstetric complications (H/OCs). Two other groups: medicated patients with documented H/OCs [hypoxia group (H-SZ (M+)] and medicated patients with documented absence of H/OCs [non-hypoxia group (NH-SZ (M+)]. The content of f-SatIII was also determined in eight post-mortem brain tissues of one SZ patient. Results: f-SatIII in human leukocyte varies between 5.7 to 44 pg/ng DNA. f-SatIII CNVs in SZ patients depends on the patient's history of H/OCs. f-SatIII CN in NH-SZ (M+)-group was significantly reduced compared to H-SZ (M+)-group and HC-group (p < 10-30). f-SatIII CN in SZ patients negatively correlated with the index reflecting the seriousness of the disease (Positive and Negative Syndrome Scale). Antipsychotic therapy increases f-SatIII CN in the untreated SZ patients with a low content of the repeat and reduces the f-SatIII CN in SZ patients with high content of the repeat. In general, the SZ (M+) and SZ (M-) groups do not differ in the content of f-SatIII, but significantly differ from the HC-group by lower values of the repeat content. f-SatIII CN in the eight regions of the brain of the SZ patient varies significantly. Conclusion: The content of f-SatIII repeat in leukocytes of the most patients with SZ is significantly reduced compared to the HC. Two hypotheses were put forward: (1) the low content of the repeat is a genetic feature of SZ; and/or (2) the genomes of the SZ patients respond to chronic oxidative stress reducing the repeats copies number.

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.

Copy Number of Human Ribosomal Genes With Aging: Unchanged Mean, but Narrowed Range and Decreased Variance in Elderly Group.

Introduction: The multi-copied genes coding for the human 18, 5.8, and 28S ribosomal RNA (rRNA) are located in five pairs of acrocentric chromosomes forming so-called rDNA. Human genome contains unmethylated, slightly methylated, and hypermethylated copies of rDNA. The major research question: What is the rDNA copy number (rDNA CN) and the content of hypermethylated rDNA as a function of age? Materials and Methods: We determined the rDNA CN in the blood leukocyte genomes of 651 subjects aged 17 to 91 years. The subjects were divided into two subgroups: "elderly" group (E-group, N = 126) - individuals over 72 years of age (the age of the population's mean lifetime for Russia) and "non-elderly" group (NE-group, N = 525). The hypermethylated rDNA content was determined in the 40 DNA samples from the each group. The change in rDNA during replicative cell senescence was studied for the cultured skin fibroblast lines of five subjects from NE-group. Non-radioactive quantitative dot- and blot-hybridization techniques (NQH) were applied. Results: In the subjects from the E-group the mean rDNA CN was the same, but the range of variation was narrower compared to the NE-group: a range of 272 to 541 copies in E-group vs. 200 to 711 copies in NE-group. Unlike NE-group, the E-group genomes contained almost no hypermethylated rDNA copies. A case study of cultured skin fibroblasts from five subjects has shown that during the replicative senescence the genome lost hypermethylated rDNA copies only. Conclusion: In the elderly group, the mean rDNA CN is the same, but the range of variation is narrower compared with the younger subjects. During replicative senescence, the human fibroblast genome loses hypermethylated copies of rDNA. Two hypotheses were put forward: (1) individuals with either very low or very high rDNA content in their genomes do not survive till the age of the population's mean lifetime; and/or (2) during the aging, the human genome eliminates hypermethylated copies of rDNA.

Symmetric dimeric bisbenzimidazoles DBP(n) reduce methylation of RARB and PTEN while significantly increase methylation of rRNA genes in MCF-7 cancer cells.

BACKGROUND: Hypermethylation is observed in the promoter regions of suppressor genes in the tumor cancer cells. Reactivation of these genes by demethylation of their promoters is a prospective strategy of the anticancer therapy. Previous experiments have shown that symmetric dimeric bisbenzimidazoles DBP(n) are able to block DNA methyltransferase activities. It was also found that DBP(n) produces a moderate effect on the activation of total gene expression in HeLa-TI population containing epigenetically repressed avian sarcoma genome. PRINCIPAL FINDINGS: It is shown that DBP(n) are able to penetrate the cellular membranes and accumulate in breast carcinoma cell MCF-7, mainly in the mitochondria and in the nucleus, excluding the nucleolus. The DBP(n) are non-toxic to the cells and have a weak overall demethylation effect on genomic DNA. DBP(n) demethylate the promoter regions of the tumor suppressor genes PTEN and RARB. DBP(n) promotes expression of the genes RARB, PTEN, CDKN2A, RUNX3, Apaf-1 and APC "silent" in the MCF-7 because of the hypermethylation of their promoter regions. Simultaneously with the demethylation of the DNA in the nucleus a significant increase in the methylation level of rRNA genes in the nucleolus was detected. Increased rDNA methylation correlated with a reduction of the rRNA amount in the cells by 20-30%. It is assumed that during DNA methyltransferase activity inhibition by the DBP(n) in the nucleus, the enzyme is sequestered in the nucleolus and provides additional methylation of the rDNA that are not shielded by DBP(n). CONCLUSIONS/SIGNIFICANCE: It is concluded that DBP (n) are able to accumulate in the nucleus (excluding the nucleolus area) and in the mitochondria of cancer cells, reducing mitochondrial potential. The DBP (n) induce the demethylation of a cancer cell's genome, including the demethylation of the promoters of tumor suppressor genes. DBP (n) significantly increase the methylation of ribosomal RNA genes in the nucleoli. Therefore the further study of these compounds is needed; it could lead to the creation of new anticancer agents.

GC-Rich DNA Fragments and Oxidized Cell-Free DNA Have Different Effects on NF-kB and NRF2 Signaling in MSC.

It has been established that cell-free DNA circulating in the bloodstream affects cells. The characteristics of cfDNA depend on the physiological state of the organism. As we showed previously, diseases can cause either GC-enrichment of the cell-free DNA pool or its oxidation. Thus, in cases of cerebral atherosclerosis, heart attack and rheumatic arthritis the cell-free DNA pool is GC-enriched and, in the case of cancer, both GC-enriched and oxidized. Herein we investigated the time-dependent effect of oxidized and GC-rich cell-free DNA on NF-kB and NRF2 signaling pathways in human mesenchymal stem cells and showed that they affect cells in different ways. Oxidized DNA drastically increases expression of NRF2 in a short period of time, but the effect does not last long. GC-rich DNA causes a prolonged increase in mRNA levels of NF-kB and NRF2 which lasts 48 and 24 h, respectively.