Satellite III (1q12) Copy Number Variation in Cultured Human Skin Fibroblasts from Schizophrenic Patients and Healthy Controls

Background : The chromosome 1q12 region harbors the genome’s largest pericentromeric heterochromatin domain that includes tandemly repeated satellite III DNA [SatIII (1)]. Increased SatIII (1) copy numbers have been found in cultured human skin fibroblasts (HSFs) during replicative senescence. The aim of this study was to analyze the variation in SatIII (1) abundance in cultured HSFs at early passages depending on the levels of endogenous and exogenous stress. Methods : We studied 10 HSF cell lines with either high (HSFs from schizophrenic cases, n = 5) or low (HSFs from healthy controls, n = 5) levels of oxidative stress. The levels of endogenous stress were estimated by the amounts of reactive oxygen species, DNA damage markers (8-hydroxy-2′-deoxyguanosine, gamma-H2A histone family member X), pro-and antioxidant proteins (NADPH oxidase 4, superoxide dismutase 1, nuclear factor erythroid 2-related factor 2), and proteins that regulate apoptosis and autophagy (B-cell lymphoma 2 [Bcl-2], Bcl-2-associated X protein, light chain 3). SatIII (1) copy numbers were measured using the nonradioactive quantitative hybridization technique. For comparison, the contents of telomeric and ribosomal RNA gene repeats were determined. RNASATIII (1 and 9) were quantified using quantitative Polymerase Chain Reaction (PCR). Results : Increased SatIII (1) contents in DNA from confluent HSFs were positively correlated with increased oxidative stress. Confluent cell cultivation without medium replacement and heat shock induced a decrease of SatIII (1) in DNA in parallel with a decrease in RNASATIII (1) and an increase in RNASATIII (9). Conclusions : During HSF cultivation, cells with increased SatIII (1) content accumulated in the cell pool under conditions of exaggerated oxidative stress. This fraction of cells decreased after the additional impact of exogenous stress. The process seems to be oscillatory.

SatIII (1) transcription from the 1q12 region in various conditions has been much less studied than SatIII (9) transcription. At the same time, the 1q12 chromosomal region harbors the human genome's biggest site of heterochromatin, which consists of SatII and SatIII tandem arrays. Enukashvily et al. [34] were the first to show that SatIII (1) is decondensed, demethylated, and transcribed in senescent cells and A431 epithelial carcinoma cells, in contrast to SatIII (9). It has been also shown that the ratio of the SatIII (1)/SatIII (9) transcript in radiation-exposed cells depends on the ionized radiation (IR) dose [35].
Unlike the SatIII transcription process, copy number variation (CNV) of SatIII repeats in human genomes under various conditions has been poorly studied. Changes in satellite repeat copy numbers are often found in cancer cells  Index  SZ1 SZ2 SZ3 SZ4 SZ5 K1 K2 K3 K4 K5   1  Age  28  39  42  50  25  35  42  49  39  27  2  Sex  m  m  m  m  m  f  f  m  m  f  3 Age of SZ onset 14 16 17 18 12 n/a n/a n/a n/a n/a 4 Age of SZ manifestation 15 16 19 19 12 n/a n/a n/a n/a n/a 5 Relatives with SZ + -+ + + ----- 6 Taking antipsychotics + + + + + n/a n/a n/a n/a n/a F, Female; M, Male; n/a, Not applicable; #, line number; +, yes; -, no. [36,37]. Bersani et al. [38] presumed that the transcription of satellite DNAs induced by different environmental stress conditions could be coupled to new satellite repeat insertions and changes in the repeat copy numbers. Based on the example of SatII CNV in cancer cells, the mobile nature of satellite DNAs has been suggested [38].
We previously observed changes in SatIII (1) CNVs in mesenchymal stem cells exposed to radiation [35], cultured human skin fibroblasts (HSFs) during replicative senescence and oxidative stress [39], and blood cells from schizophrenic (SZ) patients during antipsychotic treatment [40]. The HSF pool at early passages is heterogeneous with respect to SatIII (1) quantity in cell genomes. Cells with high SatIII (1) content have low proliferative capacity and produce small clones during division. The fraction of these cells increases during replicative senescence [39]. Cells with high SatIII (1) content do not respond to proliferative stimuli and die when exposed to such impacts as inducing an adaptive response in normal cells with low SatIII (1) content. The increase in SatIII (1) content, as observed during the course of senescence, is positively correlated with telomere shortening [39].
The aim of this study was to analyze SatIII (1) CNVs in cultured HSFs at early passages. First, we determined if and how the changes in SatIII (1) content during HSF cultivation at early passages depend on the levels of endogenous oxidative stress in the cell pool. The level of oxidative stress can be assessed by a reactive oxygen species (ROS) assay, by the amount of DNA damage markers (8oxo-2′-deoxyguanosine [8-oxodG] [41,42]) and phosphorylated serine 139 histone H2A histone family member X (H2AX) (γ-H2AX) [43] in the cells. The quantitative and qualitative composition of ROS depends on the level of prooxidative (NADPH oxidase 4 [NOX4] [44] and superoxide dismutase 1 [SOD1] [45]) and antioxidative (nuclear factor erythroid 2-related factor 2 [NRF2] [46,47]) proteins. ROS can induce cell death by altering gene activity that regulates apoptosis (B-cell lymphoma 2 [Bcl-2] and Bcl-2associated X protein [BAX] [48,49]) and autophagy (light chain 3 [LC3] [50]). For comparison, along with SatIII (1) content, we monitored the contents of two more kinds of genomic tandem repeats: telomere repeat (TR) and ribosomal repeat (rDNA) in the DNA isolated from the cells. Second, we determined if there was an association between the SatIII (1) content and SatIII (1) transcript amount in the HSFs under stress induced by the long-term growth of confluent cells without medium replacement or by heat shock. We also compared the transcript levels of the two SatIII fractions (1q and 9q) during normal culture growth and under stress.

Participants
Primary adult HSFs from healthy controls (n = 5) and SZ patients (n = 5) were obtained from the Research Centre for Medical Genetics. A description of the five healthy subjects and the five SZ patients, who were the primary donors of skin cells, is presented in Table 1. The examination was conducted in the round-the-clock inpatient units of N.A. Alekseev Clinical Psychiatric Hospital No. 1 (Moscow, Russia), where the patients received standard psychiatric care. The skin samples were obtained from SZ cases with a long-term continuous form of schizophrenia. Each patient had been taking standard antipsychotics for several years (e.g., haloperidol, aminazin, risperidone, seroquel, olanzapin). Four of the five patients had nearest relatives with the same disease.
The control skin sample donors were staff members of the Research Centre for Medical 100 Genetics. They were apparently healthy, took no pharmaceuticals, and had no mental problems or relatives with mental disorders.

Cell Cultures
The cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA), 2 mM glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin at 37°C in a 5% CO 2 incubator. All of the experiments were performed using cells from passage 4. Cultures with less than 2% of cells in the S phase were considered confluent. In general, cells reached confluence after 3-4 days of plating. This was marked as Day 1 of the experiment.

Ethical Approval for the Use of Cultured Human Cells
The study was carried out in accordance with the latest version of the Declaration of Helsinki and was approved by the Independent Interdisciplinary Ethics Committee on Ethical Review for Clinical Studies (Protocol No. 4 dated March 15, 2019 for the scientific minimally interventional study "Molecular and neurophysiological markers of endogenous human psychoses"). All participants provided written informed consent to participate in the study after the procedures had been completely explained.

DNA Isolation from HSFs
To isolate the DNA, we used a standard method as previously described [51]. Briefly, 2 mL of the solution (2% sodium lauryl sarcosylate, 0.04 M EDTA, and 150 µg/mL RNAse A; Sigma, St. Louis, MO, USA) was added to the cell mass for 45 min (37°C) and then treated with proteinase K (200 µg/mL; Promega, Madison, WI, USA) for 24 h at 37°C . The lysates were extracted with an equal volume of phenol, phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1). DNA was precipitated by adding 1/10 the volume of 3 M sodium acetate (pH 5.2) and a 2.5-fold volume of ice-cold ethanol. DNA was collected by centrifugation (10,000 ×g for 15 min at 4°C), washed with 70% ethanol (v/v), and dissolved in water. DNA concentration was measured in two steps, as described earlier [51]. The final DNA quantification was performed fluorometrically using PicoGreen dsDNA Quantification Reagent (Invitrogen, Carlsbad, CA, USA). The DNA concentration in a sample was calculated according to a standard curve for DNA. We used the Enspire Multimode Microplate Reader (PerkinElmer, Turku, Finland) at λ ex = 488 nm and λ em = 528 nm.

Nonradioactive Quantitative Hybridization
The nonradioactive quantitative hybridization (NQH) method is based on the complementary hybridization of DNA immobilized on a filter with a biotin-labeled DNA probe. NQH was used for the quantification of f-SatIII, TR, and rDNA repeats, as previously detailed without modification [52,53] and Supplements of [39]. Five reference DNA probes with a known repeat content were applied to the same filter. After hybridization and biotin detection using the streptavidin-alkaline phosphatase conjugate (BCIP and nitro blue tetrazolium substrates), the filter was scanned and the spot signal intensity was measured using a customized software pack. A calibration curve was constructed, reflecting the dependence of the signal intensity on the genome's repeat copy number. The relative standard error for NQH was 5 ± 2%. The major overall error of the experiment was contributed to by the step of isolating DNA from the cells. The total standard error was 11 ± 7%. The DNA probe for SatIII (1) quantification was a 1.77-kb cloned EcoRI fragment of human satellite DNA [1] labeled with biotin-11-dUTP using nick translation. Dr. Cook (Medical Research Council, Edinburgh, UK) kindly supplied the human chromosome lql2-specific repetitive satellite DNA probe pUC1.77. The DNA probe for rDNA quantification contains rDNA sequences (5836 bp) cloned into an EcoRI site of the pBR322 vector. The rDNA fragment covered posi-tions from -515 to 5321 of the human rDNA (GenBank accession No. U13369). DNA Probe used for the detection of the human TR was biotin-(TTAGGG) 7 . Syntol (Moscow, Russia) performed the synthesis and biotin labeling of the oligo probe.

ROS Assay
The fluorescent assay was applied for ROS quantification in HSFs in the format of a 96-well tablet reader at λ ex = 488 nm and λ em = 528 nm (Enspire, PerkinElmer, Turku, Finland). The medium was replaced with 5 µm H2DCFH-DA (Molecular Probes/Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in PBS, and a fluorescence intensity was measured at 37°C. Eight (4 × 2) measurements were made for each DNA sample and 16 for the control. The slope of the line (k), which reflects the dependence of the fluorescence intensity on time, characterizes the ROS content in HSFs. This method allows quantification of the total ROS amount in HSFs (intracellular) and in the extracellular medium. For the intracellular ROS assay (i.e., only in the cells), FCA was applied. The medium was replaced with 5 µm H2DCFH-DA in PBS for 1 h at 37°C. Prior to FCA, cells were washed with Versene solution and treated with 0.25% trypsin under light microscope observation. Then the cells were transferred to Eppendorf tubes, washed with PBS, centrifuged, and resuspended in PBS.

Quantitative Polymerase Chain Reaction
Isolation of the RNA was performed using the RNeasy Mini kit (Qiagen, Hilden, Germany). RNA was quantified using the Quant-iTRiboGreen RNA Reagent (Mo-BiTec, Göttingen, Germany) on a multimode plate reader (EnSpire; PerkinElmer) at λ em = 487 nm, λ fl = 524 nm. The RNA samples were further treated with DNase I to remove possible trace amounts of DNA. After DNAse I treatment, RNA samples were reverse transcribed using the Reverse Transcriptase Kit (Sileks, Moscow, Russia). PCR was conducted with specific primers and SYBR Green intercalating dye on the StepOnePlus Real-Time Polymerase Chain Reaction (PCR) System (Applied Biosystems, Foster City, CA, USA). The primers were selected and synthesized by Evrogen (Moscow, Russia). The internal standard was the TATA box-binding protein gene. For the RNASATIII assay, primers described in [34] were applied. In each series of experiments, RNA samples treated with DNase I were used as a negative control. The PCR reaction mixture in a volume of 25 µL consisted of 2.5 µL PCR buffer (700 mmol/L Tris-HCl, pH 8.6); 166 mmol/L ammonium sulfate, 35 mmol/L MgCl 2 , 2 µL of 1.5 mmol/L dNTP solution; and 1 µL of 30 pmol/L primer solution, cDNA. PCR conditions were chosen individually for each primer pair. After denaturation for 4 min at 95°C, 40 amplification cycles were performed in the following order: 94°C for 20 s, 56-62°C for 30 s, 72°C for 30 s, and 72°C for 5 min. The data were processed using a calibration plot with a resultant error of 2%.

Fluorescence Microscopy
The Axio Scope.A1 microscope (Carl Zeiss, Oberkochen, Germany) was used for fluorescence microscopy. For γH2AX detection, cells were washed three times with PBS, fixed for 20 min in 3% PFA at 4°C, washed with PBS, and incubated for 4 h at 4°C with γH2AX-FITC antibodies. After washing with 0.01% Triton X-100 in PBS, HSFs were washed with PBS and stained with 2 µg/mL DAPI.
For fluorescence in situ hybridization (FISH), the HSFs in slide flasks were washed with PBS. The slides were removed and placed for 10 min into a cold fixation solution. After the procedure was repeated three times, the slides were dried and subjected to FISH. Before hybridization, the slides were treated with RNAse A (100 µg/mL). Hybridization was conducted using the protocol and solutions from Abbott Laboratories (Abbott Park, IL, USA) in the Statspin ThermoBrite Slide Denaturation and Hybridization System (Abbot Molecular Inc., Des Plaines, IL, USA) at 42°. Nuclei were stained with propidium iodide. The f-SatIII FISH probe was a 1.77-kb-cloned EcoRI fragment of human satellite DNA [54]. Labeling of plasmid pUC1.77 was performed using nick translation with the CGH Nick Translation Kit (Abbott Molecular) according to the manufacturer's protocol.

Statistical Analyses
Each test was repeated in triplicate. In FCA, the medians of the signal intensities were analyzed. The significance of the observed differences was analyzed using the nonparametric Mann-Whitney test (p) and Kolmogorov-Smirnov test (D and α). Spearman analysis (Rs, p) was used to analyze correlations between the two parameters. p < 0.01 and α < 0.04 were considered statistically significant.

Results
The HSFs of healthy controls (hc-HSFs, K1-K5) and SZ patients (sz-HSFs, SZ1-SZ5) were studied. After inoculation, sz-HSFs and hc-HSFs were cultured until confluence (3-4 days). Cultures with less that 1% of cells in the S phase were considered confluent. This was marked as Day 1 (control) of the experiment. Fig. 1 shows the content data of the three tandem repeats (SatIII (1), TR, and rDNA) in DNA isolated from cells that had reached confluence (control). For reference, the contents of the repeats in actively proliferating cell culture 2 days before the confluent state (-2 days) were also given.
The TR content in DNA varied from 71 pg/µg DNA (1SZ) up to 335 pg/µg (4K) (Fig. 1A,a2), corresponding with the average LTL, approximately 2 to 7 kb. Hc-HSFs carried more copies of TR (D = 0.8, α = 0.036; p < 0.01) in their genomes than sz-HSFs, suggesting longer telomeres in hc-HSFs. A comparison of confluent cell DNA (control) to DNA isolated from actively proliferating cells (-2 days) showed a slight decrease of TR contents in DNA of confluent sz-HSFs. The TR content in DNA of the confluent HSFs negatively correlated (Fig. 1A,a4) with the SatIII (1) content (Rs = -0.88, p < 0.001, n = 10).  The numbers of rDNA copies in HSFs varied from 386 (3SZ) up to 454(5K). Sz-HSFs and hc-HSFs did not differ by rDNA abundance in the cellular DNA (Fig. 1A,a3). This index did not change during cell cultivation. Thus, in the process of culturing HSFs to a confluent state, the SatIII (1) content in the isolated DNA increases. One of the possible reasons for the SatIII (1) content increase may be an increase in the amount of RNA SATIII (1).
Thus, it was shown that the content of the SatIII (1) repeat in DNA isolated from confluent cells correlated with the content of RNASATIII in RNA isolated from the same cells. Stress is an inducer of SatIII (1) transcription [34,35]. To determine the level of endogenous stress that could induce satellite transcription and subsequent satellite DNA amplification, we determined some characteristics, which indicate the level of oxidative stress in the HSF pool.

Cellular DNA Damage
To estimate the double-strand DNA breaks rate in nuclear DNA, antibodies to 301 phosphorylated form of histone Н2АХ (γH2AX) [43] were applied ( Fig. 2A,a1). For the reader's convenience, a median value of cell signal for each HSF cell line was normalized to the maximum median value of the signal in the sample (parameter γH2AX = m[FL-γH2AX]i/m[FL-γH2AX]max). The amounts of γH2AX in sz-HSFs were higher than those in hc-HSFs (D = 0.8, α = 0.036; p < 0.01). The maximum damage degree was observed for SZ1, whereas the minimum one was observed for K4 ( Fig. 2A,a2).
An analysis of the dependence of γH2AX on FSC parameter, which indicates the cell size, allowed us to distinguish the cell subset R1 (Fig. 2C,c1-c3 and 2E,e1) with large sizes and relatively high, compared to the other cells, level of the DNA damage marker. This fraction varied in the confluent HSFs from 6% to 16% and correlated positively with SatIII (1) repeat content in the DNA (Rs = +0.82, p = 0.0038, n = 10), while correlated negatively (Rs = -0.84, p = 0.0022, n = 10) with the TR abundance (Fig. 2D,d1,d2). The rDNA abundance in the genome was not associated with the size of R1 fraction (Fig. 2D,d3).
FISH was conducted to analyze the state of SatIII (1) repeat (Fig. 2E,e2). SatIII (1) is localized in two homologous sites of the nucleus. In cells of normal size, they are visualized as two compact spots. In large cells, the SatIII (1) signal area is much larger, suggesting both chromatin decondensation and an increased total copy number of the repeat in such cells.
Thus, the oxidative modification seems to be the major cause of DNA breaks, resulting from the high content of endogenous ROS in HSF cell pools. The ROS amount depends on the balance of pro-and antioxidant enzyme activity. We assayed the ROS amount and the contents of three proteins (NOX4, SOD1, and NRF2) that regulate ROS abundance in the cell.

ROS Abundance in Confluent HSFs
The ROS were quantified using H 2 DCFH-DA chemical reagent and a tablet reader (Fig. 3A). To estimate the ROS amount, the synthesis rate constant DCF (k) was determined in confluent sz-HSFs after the reagent had been added. The k value for sz-HSFs was considerably higher than that for hc-HSFs, suggesting a higher ROS level in the cells derived from SZ patients (Fig. 3A,a1,a2).
The distribution of unfixed cells by ROS amount was estimated using FCA (Fig. 3A,a3). The HSF cell pool appeared to be heterogeneous by this parameter. Three fractions could be distinguished. R1 were large cells with an elevated ROS content. R2 and R3 were cells of approximately the same normal size, but with different ROS levels.
The mRNA and protein levels of SOD1 (SOD1 index) were the same in sz-HSFs and hc-HSFs (p > 0.1; Fig. 3D). К5 line cells contained the maximum level of the protein. The SOD1 index did not correlate with RNASOD1 amount.

Apoptosis and Autophagy Gene Expression
We found no significant differences in the RNA and protein levels of BAX and BCL2 between sz-HSFs and hc-HSFs ( Fig. 4A-C). The maximum BAX index was observed for SZ1, whereas the minimum one was observed for K3 and K4 (Fig. 4A,a3). A BAX/BCL2 ratio, which represents the apoptosis intensity [55], was higher for sz-HSFs than for hc-HSFs (p < 0.015), with the exception of the К5 cell line. For К5, high values of RNABAX /RNABCL2 and BAX/BCL2 indices were observed (Fig. 4C).
The large size cell fraction (R1) was enriched with cells with a high level of LC3 protein (Fig. 4E,e1,e2). A joint analysis of levels of γH2AX and LC3 in the same cells  showed that the process of autophagy was activated in cells with a high DNA damage degree, which were part of fraction R1 (Fig. 4E,e3). Fig. 5 shows the dependence of SatIII (1) content on the abovementioned characteristics of the cell lines. The SatIII (1) repeat abundance was positively correlated with the levels of DNA damage markers (γH2AX and 8-oxodG), proteins that modulate ROS amount (NOX4 and NRF2), and indices that represent the apoptosis and autophagy intensity (BAX and LC3). The largest SatIII (1) copy number, as measured for DNA derived from SZ1 cells, corresponds to the maximum values of the parameters that display the level of oxidative stress. A positive correlation between the stress indicators and the content of the SatIII (1) repeat in DNA was also preserved in the individual groups (Fig. 5). Thus, we showed that a high SatIII (1) content in cell DNA is associated with exaggerated endogenous oxidative stress in the cell population.

Confluent Cell Cultivation without Medium Replacement
A 4-day cultivation of confluent HSFs without medium replacement was followed by a decrease of SatIII (1) repeat content in the DNA of all sz-HSFs by a factor of 1.5 to 4, with the maximum effect observed for SZ1 (Fig. 6A,a1,a3). In hc-HSFs, SatIII (1) copy numbers did not reduce (К1) or reduced them by a factor of 1.1 to 1.6 (i.e., to a lesser degree than in szHSFs). Unlike SatIII (1) repeats, the abundance of TR in HSF DNAs was increased or remained unchanged (К1). Long-term HSF growth without medium replacement was accompanied by no changes in rDNA content (Fig. 6B,b1). We explored changes in RNASATIII amount after 2 days of confluent cell growth. The RNASATIII (1) amount in RNA isolated from the cells increased by a factor of 1.2 (K2) to 140 (K3) (Fig. 6C,c1,c3). By contrast, the RNASATIII (9) amount decreased by a factor of 1.5 (SZ5) to 6 (K2) (Fig. 6D,d1,d3). The RNASATIII (1)/RNASATIII (9) ratio increased by a factor of 2 (SZ5) to 400 (K3). Thus, long-term confluent cell cultivation induced a decrease in the SatIII (1) content in DNA together with a simultaneous increase in the amount of RNASATIII (1) in RNA isolated from the cells.

Discussion
The aim of this study was to analyze the variation in SatIII (1) abundance in cultured HSFs at early passages depending on the levels of endogenous and exogenous stress. To this end, we selected HSFs from healthy individuals and HSFs from chronic SZ patients. We chose sz-HSFs because it is known that patients with schizophrenia experience systemic oxidative stress [56]; therefore, the cells of SZ patients should potentially have a higher level of ROS. This assumption was confirmed, as sz-HSFs produced more ROS after they reached confluence, compared to cells from healthy skin donors (Fig. 3A). A higher ROS level in sz-HSFs correlated with a higher degree of DNA damage (Fig. 2) and higher contents of the pro-oxidant protein NOX4 (Fig. 3), proapoptotic protein BAX, and protein LC3 involved in the autophagy process (Fig. 4). Previously, elevated NOX4 protein is detected in the blood lymphocytes and brain cells of SZ patients [57,58]. The DNA damage degree is also increased in both the primary and cultured cells of SZ patients [59]. The process of apoptosis is more active in SZ patients than in healthy controls [60].
Thus, we studied two groups of HSFs with different levels of endogenous ROS. The high level of ROS in the sz-HSFs is apparently due to the genetic characteristics associated with the development of the disease. In addition, patients had been taking antipsychotics for a long period of time, which could potentially cause epigenetic changes in skin cells that caused increased levels of stress or/and high SatIII (1) content. However, there are no data in the literature on the effects of antipsychotics in vivo on cultured skin cells outside the body. By contrast, fibroblasts are considered a convenient model that allows the assessment of genetic changes in the absence of endogenous influence of the body's environment, including the influence of therapy [61].
We analyzed the effects of three types of cell culture conditions on the repeat contents in cellular DNA.

Effects of Endogenous ROS Levels on SatIII (1) CNVs in HSFs
A high level of oxidative stress and DNA damage markers in HSFs was associated with increased SatIII (1) repeat content in the DNA isolated from the confluent cells (Fig. 5). An analysis of histograms of cell distribution by γH2AX marker content showed that many cells of sz-HSFs had a high marker content, but especially increased γH2AX was observed in a small subset of large-size cells. The frac-tion of such cells was correlated with the SatIII (1) content in the total DNA isolated from the cells (Fig. 2). The data obtained using FISH suggested that in these cells, the SatIII (1) repeat copy numbers were increased and/or chromatin decondensation occurred at the site of hybridization with a probe for SatIII (1).
A high SatIII (1) content in total DNA was correlated with a low TR content (Fig. 1). Earlier, opposite changes of TR and SatIII (1) contents in the cellular DNA were revealed when studying variation of these tandem repeats during replicative senescence of five HSF lines [39], in various brain regions of a SZ patient [62] and in the research of the human blood cell response for an affective stress [63]. In every case, increased SatIII (1) content in the cellular DNA was linked to a reduced TR count.
For the SatIII (1) repeat region, DNA decondensation and demethylation were previously found at late passages, accompanied by an exaggerated transcription of SatIII (1) DNA [34]. We measured the RNASATIII (1) levels in HSFs (Fig. 1). The transcript amount in sz-HSFs exceeded that in hc-HSFs, correlating with a higher SatIII (1) repeat content in sz-HSFs. An increased level of satellite transcription under conditions of endogenous oxidative stress may be one of the reasons for the increase in the content of the SatIII (1) repeat in some cells of the population.
One report [34] also showed that senescence was not accompanied by the transcription of SatIII (9) repeats localized in the pericentromeric region of chromosome 9, in contrast to SatIII (1). An assay of RNASATIII (9) showed that the amount of the transcript was decreased in sz-HSFs compared to hc-HSFs. The activation of SatIII (1) transcription was accompanied by a decrease in SatIII (9) transcript level (Fig. 1B,b4). We previously observed a similar phenomenon when studying the effects of cell exposure to ionizing radiation [35].
In total, our findings suggest that under the stress caused by endogenous factors, an increase in SatIII (1) copy numbers occurs in cells with a low count of TRs. Seemingly, these cells undergo the biggest ROS impact. Telomere shortening is associated with heterochromatin decondensation and activation of transcription from these regions. A mechanism previously proposed by Bersani et al. [38] may be involved in the increase of SatIII (1) cellular content under the action of stress factors. By means of reverse transcriptase, a DNA chain is synthesized from RNASATIII, and the DNA-RNA hybrids then forms, inserts into the transcribed region, and undergoes the action of DNA repair enzymes. The region 1q12 under exploration is known to be one of the most unstable chromatin sites [63]. In this chromatin region, the double-strand break repair processes are suppressed, thus increasing the chances of successful integration of the DNA-RNA hybrids into DNA [64].
The insertion of additional SatIII (1) fragments into the heterochromatin regions in some cells cannot be detected by analyzing the size of the chromatin block on metaphase chromosomes. We previously showed that cells with an elevated SatIII (1) content are unable to respond to various impacts that require genome architecture rearrangements necessary for changing the gene expression profiles in response to stress. In particular, these cells do not respond to proliferative stimuli (i.e., cannot enter the mitosis phase). In addition, such cells fail to launch an adaptive response for an exposure to small doses of ionizing radiation [35] and perish as a result of even a weak toxic impact.
The correlation of SatIII (1) copy number with stress markers and sufficiently high SatIII (1) transcription level in HSFs suggests that even relatively young cell pools (at early passages) can accrue cells with high SatIII (1) content during cultivation, which do not divide and are not resistant to stronger types of stressor impacts. To prove this assumption, we analyzed the changes of SatIII (1) content caused by two types of stress.

Cultivation of Confluent HSFs without Medium Replacement
Cultivating cells for a long time without medium replacement is accompanied by stress resulting from a decline of nutrients in the medium and cellular debris accumulation due to apoptosis, which can induce extra ROS synthesis in living cells [65]. Four days after HSFs had reached confluency, the SatIII (1) copy numbers significantly decreased in DNA samples isolated from sz-HSFs (Fig. 6A,a1,a3). A slight decrease of SatIII (1) content also occurred in four DNA samples isolated from hc-HSFs. Simultaneously with the decrease of SatIII (1) content, an increase in TR abundance was observed in DNA isolated from HSFs (Fig. 6B,b2). The copy numbers of ribosomal repeat remained virtually unchanged in the isolated DNA (Fig. 6B,b1). The observed changes in the contents of the two repeats in DNA isolated from the cells after long-term growth without medium replacement suggest that this stress type induces death of cells with high SatIII (1) content and relatively low TR abundance in the pools of HSFs. These findings corroborate the reports of hypersensibility of cells with high SatIII (1) contents to damaging impacts due to a blockage of the adaptive response in these cells [35].

Heat Shock Effect
The cell's response to heat shock considerably differs from the its response to prolonged cultivation without medium replacement by the RNASATIII (1)/RNASATIII (9) ratio change patterns. Heat shock induced SatIII (9) transcription, as previously described [14], while the resultant amount of SatIII (1) transcripts was considerably reduced ( Fig. 6C-E).
Despite the difference in the changes of transcription of different SatIII fragments under heat shock and cultivation without medium replacement, the patterns of changes in SatIII (1) and TR copy numbers in the cellular DNA were similar: while SatIII (1) repeats were reduced, the TR ar-rays were enlarged (Fig. 6B). These data suggest a stressinduced process of death in cells with a high abundance of SatIII (1), and simultaneously, a low content of TR.

Satellite III CNV in Cultured HSFs
An integrated analysis to summarize the previous reports [35,39] and the data obtained in this study suggested a scheme of satellite III (1q12) CNV in cultured cells with different levels of endogenous ROS production (Fig. 7).
The cell pool of HSFs is heterogeneous by the copy numbers of satellite III (1q12) repeats in the cell genomes. As early as the first passages, they contain a small fraction of cells with an elevated SatIII (1) content [39]. The clones with high SatIII (1) content consist of a few cells, because the proliferative capacity of these cells is considerably abated as compared to cells harboring low SatIII (1) abundance in the genome. The amounts of TR in cells that had formed clones (dividing cell) with high and with low SatIII (1) contents did not differ [39].
The cell pool of HSFs is also heterogeneous by ROS level (Fig. 3A). In confluent HSFs, cells with high ROS content formed a subset, which was characterized by a large cell size and high DNA damage degree. The size of this cell fraction correlated with increased SatIII (1) content and decreased TR content in the DNA isolated from the whole bulk of cells. FISH data suggested that SatIII (1) content was increased, for the most part, in the cells that belonged to this fraction (Fig. 2). As previously shown, cells with the same characteristics accrued during replicative senescence [39].
An extra stress impact induces death of the cells with a high SatIII (1) content and a low TR count. Such cells are not capable of development of the adaptive response and have hypersensibility to DNA damaging agents [35]. The abovementioned process occurs during various stress types -cultivation without medium replacement and heat shock (our study's findings), as well as exposure to ionization radiation [35]. As a result, the after-stress DNA isolated from HSFs had a decreased SatIII (1) content, and to a lesser degree, an increased TR content (Fig. 6). Perhaps, the cells with high SatIII (1) content and low TR content dying under exposure to small radiation doses, are a source of extracellular DNA fragments, which induce a bystander effect and an adaptive response in the other (surviving) cells of the cell pool. Existence of such a subset in every cell pool was hypothesized earlier [66].
In vivo, the body inevitably undergo the environmental stress impact, sooner or later. Thus, the SatIII (1) content in the total DNA passes a cycle of an increase and a decrease, and the TR content passes a similar, but phaseshifted cycle, as shown in Fig. 7. The figure shows one oscillation, but provided that cell pools are regularly renewed in a living body and the stresses are also repeated from time to time all over the lifetime, one can hypothesize that the oscillations are recurrent in vivo. This speculation had be- come the basis for a 'pendulum' model of SatIII (1) dynamics we proposed early [53] and corroborated in the present study.
The stress evokes an increase of RNASATIII (1) amount in HSFs during normal growth (Figs. 1,6). Transcription of the two fractions of satellite III, localized on chromosome 1 and chromosome 9, depends on the stress type. A high ROS level during cultivation induces an augmentation of RNASATIII (1) transcripts and a decrease of RNASATIII (9) transcripts. By contrast, a heat shock induces an augmentation of RNASATIII (9) amount and a decrease of RNASATIII (1) amount by several times (Fig. 6). As shown earlier, decondensation and transcription of heterochromatin during replicative senescence (when oxidative stress is increased) were observed for SatIII (1) fragment only, but not for SatIII (9) fragment [34].
The different patterns of stress response found in the two satellite III fragments seem to be associated with dif-ferent mechanisms of transcription activation in these chromatin regions. For SatIII (9), a transcription inducer is heat shock factor [31][32][33]. A factor that triggers SatIII (1) transcription under oxidative stress and blocks transcription under heat shock is still unknown.
A bulk of data obtained in recent decades has shown that SatIII (1) repeat CNV in the same cell pool is a process typical for each type of cultivated and primary cells. The augmentation of SatIII (1) copy numbers after a stress impact of medium intensity and the decline after a stronger impact was earlier observed for cultivated human blood lymphocytes, mesenchymal stem cells, and skin fibroblasts [35,39]. Similar changes were observed in healthy human blood leucocytes under an affective stress [52]. On the peak of stress development, SatIII (1) copy number increased and TR copy number decreased in the leucocyte DNA. After the stress reverse changes were observed in the contents of the two types of DNA repeats. The same regularity was observed in the cells from various human brain regionsa negative correlation between SatIII (1) and TR contents. A chronic stress favors elimination of the cells with high SatIII (1) content from blood in vivo. Perhaps that is why blood leucocyte DNA isolated from SZ patients contained low copy numbers of SatIII (1) [62].
We believe that the increase in SatIII (1) content in some cells of the pool is a manifestation of a general mechanism that is aimed at division arrest in cells with damaged chromatin. A high content of SatIII (1) in the cell is associated with blocking changes in the chromatin structure in response to proliferative and other stimuli. Such cells are subject to being eliminated from the pool; the more actively, the higher the stress in the body. At a relatively low level of stress, these cells accumulate in the pool during senescence. These cells are harmful ballast, as they can disrupt the functional activity of the entire cell population. This is especially true for cells of the nervous tissue that are involved in signal transmission along the cell chain.
It remains unknown what causes the arrest of chromatin mobility under stress conditions, and we do not consider the large size of SatIII (1) as the major cause of arrest. Other satellite chromatin fragments seem to be also involved in a similar scheme of CNVs. This mechanism warrants further research. First, answers to the following questions should be obtained.
(1) How does stress increase the size of the SatIII (1) repeat block in some cells of a normal (non-cancer) cell pool? The transcription from the satellite repeats is launched in most cells of the pool, but just a small fraction of the cells demonstrates an increased abundance of the repeat. One can assume that these cells may have an elevated level of reverse transcriptase, or their 1q12 region contains an increased number of chromatin break sites, where the hybrid molecules can be inserted.
(2) Are there any CNVs of other repeats, such as SatIII (9), during senescence and stress?
(3) What is the difference between the transcription activation mechanisms of the two fragments of satellite III, which are localized on chromosomes 1 and 9? Why is SatIII (1) transcription activation linked to SatIII (9) transcription suppression?

Conclusions
During human skin fibroblast cultivation, cells with increased SatIII (1q12) content accumulated in the cell pool under conditions of exaggerated oxidative stress intrinsic to schizophrenia. This fraction of cells decreased after the additional impact of exogenous stress. The process of SatIII increase/reduction seems to be oscillatory.