[The changing of cell-free DNA properties of peripheral blood and TCR-mutant cell frequency in individuals exposed to ionizing radiation].

Some properties of the cell-free DNA (cfDNA) of peripheral blood plasma were assessed in 153 employees of atomic industry enterprises. The contents of ribosomal repeat (rDNA) and its concentration in plasma increased in cfDNA of the group of persons in comparison with non-irradiated individuals. The contents of satellite III in cfDNA of donors and of irradiated persons do not differ and less than in DNA nucleus. The correlation between cumulative dose of radiation, contents of rDNA in cfDNA and the frequency of lymphocytes bearing mutations at T-cell receptor (TCR) locus was obtained. The definition of three indications in irradiated persons: the contents of ribosomal genes in cfDNA, TCR-mutant cell frequency and concentration of ribosomal genes in blood plasma--may be useful for revealing individuals in organism of which an intensive cell apoptosis takes place and there is an increased probability of carcinogenesis and of progress of disease of immune system.

[The DNA fragments obtained from the culture media exposed to adaptive doses of the ionizing radiation as factors of stress signaling between lymphocytes and bystander cells].

At the initial stages of an adaptive response the transposition of the homologous chromosome loci from the peripheral parts of the nucleus and their approach happens. It is necessary for the repair of DNA double strand breaks in the process of the homologous recombination. Was shown that the chromosome loci transposition and accompanied by the nucleolus activities took place first in the irradiated (X-rays, 10 cGy) G0-lymphocytes, and then in the intact (bystander) cells incubated in the growth medium of irradiated lymphocytes. If there is a bystander effect the quantity of irradiated cells may be three order less than the bystander cells that affirms the great capacity of stress-signalization system. Moreover, the DNA fragments (the factors of stress signaling) were obtained from the growth medium supernatant of the irradiated and of the intact lymphocytes. In other independent experiments they were inoculated into the growth medium of recipient cells. Was demonstrated that there is loci transposition of homologous chromosomes loci and of nucleus activity after introducing the DNA fragments of irradiated cells. After introducing the DNA fragments of non-irradiated cells the both effects were not observed. In the work the characteristics of the obtained factors and the possible ways of stress signaling between the irradiated and the bystander lymphocytes were discussed.

Stimulatory effect of fragments from transcribed region of ribosomal repeat on human peripheral blood lymphocytes.

Fragments from the transcribed region of the ribosomal repeat include considerable amounts of unmethylated CpG DNA motifs. These motifs activate immune cells via the interaction with Toll receptors. In vitro experiments confirmed the stimulatory effect of transcribed region of ribosomal repeat on human lymphocytes. Culturing of lymphocytes in a medium containing 2-20,000 ng/ml fragments from transcribed region of ribosomal repeat was accompanied by structural changes in the nucleus in a considerable number of cells. These changes manifested in translocation of pericentromeric heterochromatin from the membrane to the center of the nucleus and activation of the nucleolus and were accompanied by a significant increase in interleukin-6 production and slight stimulation of tumor necrosis factor-a synthesis. The transcribed region of the ribosomal repeat and E. coli DNA had various effects on quantitative parameters of lymphocytes. Our results suggest the existence of mechanisms of stimulation not mediated by the interaction of CpG DNA motifs with Toll receptors.

Blood serum DNA in patients with rheumatoid arthritis is considerably enriched with fragments of ribosomal repeats containing immunostimulatory CpG-motifs.

We previously hypothesized that the sequence of transcribed region of human ribosomal repeats is selectively accumulated in circulating extracellular DNA due to its increased resistance to double-strand breaks caused by accumulation of single-chain breaks produced by nucleases. The contents of rDNA in blood serum DNA and in DNA from leukocytic nuclei both in healthy donors and in patients with rheumatoid arthritis were compared using dot hybridization method. By the content of non-methylated CpG-repeats, transcribed region of rDNA is identical to bacterial DNA, which is characterized by potent immunostimulatory effect. The transcribed region of rDNA (13.3 kb) contains more than 200 CpG-motifs capable of interacting with TLR9 receptors, which are the mediators of the cell immune response to the action of CpG-rich DNA fragments. The data suggest that DNA from dead cells circulating in the peripheral blood is enriched with sequences possessing potent immunostimulatory properties.

[The structural reorganization of chromatin as a process of its self-organization in eukaryotic cells and DNA repair problem].

Below were demonstrated the differences in the cell reaction of the chromosome loci transference induced by adaptive doses X-radiation in the cells nucleus in norm cells and in cells with repair process defect of the oncological patients and patients with hereditary disease. It was supposed that the transference of the homologous chromosome loci is necessary for the realization of the correct DNA's double strand repair. The chromosome loci transference in normal cells could be induced by different factors such as X-radiation, RNA-polymerize II repression by alpha-amanitin and possible by other factors. In cells with BSCA1 and BRCA2 genes mutations the chromosome loci transference could not be induced by the X-radiation, but it could be induced by the RNA-polymerize II repression by alpha-amanitin. The defect of the chromosome loci transference condition on genetics or the another determinatives and this defect could be the one of the important reasons of the genome instability. There was suggested the new conception of the mechanisms of the cell differentiation and cell chronological senescence. The vector of the cell differentiation mechanisms is the progressive chromatin condensation determined by the physical mechanisms, i.e., transformation from less probable (the stem cells) to more probable cell system state (the differentiated cells) and as a result of it changing of the genes transcription. The continued chromatin condensation (most pronounced in the old cells) decreases the probability of the realization of the DNA repair as the reason of the chromosome loci transference limitation. In this work are presented experimental and theoretical information that proves our conception.

[Transposition of chromosome loci in bystander cells upon exposure to an adaptive dose of ionizing radiation].

During the process of the realization of the bystander effect the trans of the Signal from irradiated cells to the intact cell (bystander cells) happens. So both type of cells (irradiated and intact cells) have the same damages and reactions. There are new data about bystander effect as the transduction mechanism of the adaptive response and we have investigated this phenomenon. There are an incubation of the intact (bystander cells) and the exposed (X-radiation of 10 cGy) human lymphocytes and we analyze the location of the centromeric loci of the first chromosome. We observed hat for the first time that after X-ray exposition of the adaptive doses the transposition of the chromosome loci from the peripheral to the central parts of the nucleus in intact (bystander cell) G0-lymphocytes which were incubated in the growth environment cells with irradiated cells removal. We support that the starting states of the adaptive response is the loci extrication of the matrix, the transposition and the approach homologous chromosomes. This process is necessary for the DNA double strand breaks reparation (in the case of injured dose X-radiation) with the participation of the homologous recombination.

[Early and late responses to oxidative stress in human dermal fibroblasts of healthy donors and rheumatoid arthritis patients. Relationship between the cell death rate and the genomic dosage of active ribosomal genes].

A study was made of the effect of the oxidizing agent potassium chromate (K2CrO4, PC) on cultured dermal fibroblasts of a healthy donor and three patients with rheumatoid arthritis (RA). Characteristics of the rRNA gene (RG) complex-RG copy number, active RG (ARG) dosage, and 18S rRNA content--were determined for each cell line. In cells of the healthy donor, oxidative stress caused by low doses of PC (2-4 microM, 1-4 h) induced an early response, including a 50-80% increase in total RNA and rRNA. An appreciable activation of the nucleolus was observed cytochemically, by silver staining and morphometry. The early response grew considerably lower with the increasing passage number and/or PC concentration. Exposure to 6-12 microM PC for 24 h led to a progressive cell death (late response). The existence and intensity of the early response correlated positively with the cell survival during further culturing. Cells of the RA patients displayed almost no early response even at early passages: total RNA did not increase, and rRNA increased by no more than 10%. Cell disruption (apoptosis) during further culturing was more intense than in the line originating from the healthy donor. The apoptosis intensity characterized by the increase in the content of DNA fragments in the culture medium and in the caspase 3 activity, was inversely proportional to the ARG dosage in the genome. The results provide the first quantitative characterization of the early and late responses of cells to PC-induced oxidative stress and suggest a role of the ARG dosage in cell survival in stress.

[Activation of total and ribosomal RNA transcription under adapting doses of ionizing radiation inducing displacement of chromosome loci in human G0-lymphocyte]. / Aktivatsiia transkriptsii total'noi i ribosomnoi RNK pri vozdeistvii adaptiruiushchikh doz ioniziruiushchei radiatsii, indutsiruiushchikh izmenenie koordinat lokusov khromosom v iadrakh G0-limfotsitov cheloveka.

As we demonstrated earlier, the adapting X-ray doses (3 and 10 cGy) induced movement of chromosome centromeric loci in G0-lymphocyte nuclei. In the present study we investigated the influence of X-rays with 3 and 10 cGy doses on the content of total, 18S and 45S rRNA in human G0-lymphocytes because it is known that the transcription products participate in nucleus organization. It was shown that 3 h after irradiation the content of both total and 18S RNA was significantly increased. The 3 cGy dose induced higher level of the rRNA than 10 cGy dose did in cells of some individuals. At the same time, the 45S RNA content was not changed significantly. This result may suggest that process of rRNA transcription and primary transcript (45S rRNA) processing have been completed during 3 h after irradiation. The data about an activation of rRNA synthesis were confirmed by cytological observation. Under 3 and 10 cGy doses both nuclei diameter and area of the Ag-stained granules were increased, depending on dose. These data also may be connected with an initiation of rRNA transcription because of correlation of Ag-painting with nucleolus activity. Thus, adapting X-ray doses induce displacement of chromosome loci in lymphocyte nuclei and activation of rRNA transcription. Further investigations are required for understanding of these phenomena interconnection.

[The 3H-thymidine incorporation into G0 human lymphocytes induced by low doses of UVC radiation]. / Indutsiruemoe malymi dozami UVC-izlucheniia vkliuchenie 3H-timidina v G0-limfotsity cheloveka.

The 3H-thymidine incorporation in human lymphocytes of healthy donors induced by UVC radiation under doses 0.0008-60 J/m2 was investigated. It was shown that the incorporation of 3H-thymidine increases under doses in interval 0.1-20 and is constant under doses higher than 20 J/m2. Under doses in interval 0.006-0.03 J/m2 near a half of all samples had the level of incorporation increased in comparison with control samples. We connect the presence, absence or variability of this effect with individual peculiarities of cells and with different activity of cell subpopulations that are different on morphological and physiological characteristics. The hypothesis about the role of this factor in the influence of low doses of pathogenic agents (UVC and X-radiation, chemical compounds) on human lymphocytes is discussed.

[Structural and functional changing induced by exposure to adaptive doses of X-rays in the human lymphocytes both normal and defective by reparation of DNA double strands breaks]. / Strukturnye i funktsional'nye preobrazovaniia, indutsiruemye X-radiatsiei v diapazone adaptiruiushchikh doz, v normal'nykh i defektnykh do reparatsiidvoinykh razryvov DNK G0-limfotsitakh cheloveka.

In the present work it is shown that the phenomenon of interphase chromosome centromeric region displacement, earlier revealed by the authors, is not realized in G0-lymphocytes with heterozygous BRCA1/2 gene mutations. The role of these genes in DNA double strand break (DSB) reparation is known. It is concluded that chromosome locus displacement is necessary for DSB repair, at least in the process of homologous recombination. In accordance with our data, some feature (pericentromeric cluster disintegration and displacement, the nucleus size increasing) characteristic for S- and G0-lymphocytes are observed in normal G0-lymphocytes treated with 3 and 10 cGy. However, the size of nucleus in G0-lymphocytes is restored through 6 hours after irradiation in opposite to the process in dividing cells. It was proposed that some typical for resting cell functions of G0-lymphocytes after inducing by adaptive doze of radiation are stopped as similarly as after stimulation of cells. Interestingly, that the process of the induced chromosome loci displacement is correlated with the decreasing of DNA reparation possibilities under UV-irradiation. The induced apoptosis level also decreases when chromosome loci are displaced. The possible mechanisms of the revealed phenomenon are discussed. This research supported by RFBR grant (No. 01-04-49180).

[Theoretical and experimental approach to the problem of changes in functional capability of cells under the effects of adaptive doses of ionizing radiation]. / Teoreticheskie i éksperimental'nye podkhody k probleme indutsiruemykh adaptiruiushchimi dozami ioniziruiushchei radiatsii izmenenii funktsional'nykh vozmozhnostei kletok.

It is concluded that a dose range from background dose to several cGy may be separated into two parts: a) first--the interval of small doses limited from above by D* which is determined from (1 g (D*/Doc)) < -0.51 g (n/2)), where Dse is an average dose of a single event, n--quantity of irradiated cells; in this interval only one track intersects a sensitive volume; b) second--the interval of low doses, in which in average one track intersects the volume and which is ranged from top D* to bottom Dse. Because events in this region qualitatively are similar to background events, cells in the dose range b) may be adapted to the influence of radiation. The first stage of the adaptive response of cells is associated with chromosome loci (centromere) movement in a cell nucleus and as we suggest the latter is the fundamental mechanism for repairing DSB DNA and switching of gene transcription. Because the movement of chromosome loci both in the resting cells under the adapting doses and in the normal dividing cells is much the same (but the latter lose their function characteristic for differentiated resting cells), it could be assumed that the resting cells under the adapting doses also lose their functional parameters. Under chronic exposure to low doses this functional changes can be principal for discussion on the influence of low doses on health.

[Elements of structural organization of the transcribed area of the ribosomal repeat (rDNA) in human peripheral blood lymphocytes]. / Elementy strukturnoi organizatsii transkribiruemoi oblasti ribosomnogo povtora (rDNK) v limfotsitakh perifericheskoi krovi cheloveka.

The rDNA transcribed region (TR) was tested for accessibility to RsaI recognizing 15 TR sites, DNase I, and photoinducible arylazide (N-(4-azido-2-hydroxybenzoyl)-N,N'-diaminoheptane acetate) in isolated nuclei and, with arylazide, in intact cells. Arylazide entered cells well and did not appreciably affect the chromatin structure. Its photolysis products efficiently modified DNA in accessible sites. Single-strand breaks made by DNase I were not transformed in double-stranded in rDNA TR, suggesting the necessity of denaturing electrophoresis for such an analysis. About 70% of all rDNA copies proved poorly inaccessible to endonucleases and arylazide, the accessibility being higher in their 18S and 5.8S rRNA gene regions than in the regions of the external transcribed spacers (ETSs) and the 28S rRNA gene. Proteinase K disrupted this structure, and the corresponding copies were extracted from nuclei. This explained why in situ hybridization occasionally fails to reveal rDNA in the nucleolar fibrillar center (FC) on electron microscopic preparations. In other rDNA copies, TR (excluding 5'-ETS) was accessible to nucleases and arylazide. These copies were not extracted from nuclei treated with proteinase K. Some of their RsaI sites were protected by tightly bound proteins. Seven such regions were identified in TR. Possible association of the molecular structure, nucleolar location, and functional state of rDNA is discussed.

[The accumulation of single-stranded breaks does not lead to paired DNA damage--the characteristic of the transcribing fragment of the human ribosomal operon that allows its being detected in biological fluids at the death of different body cells]. / Nakoplenie odinochnykh razryvov ne provodit k parnomu razryvu DNK--osobennost' transkribiruemogo fragmenta ribosomnogo operona cheloveka, pozvoliaiushchaia ego detektirovat' v biologicheskikh zhidkostiakh pri gibeli razlichnykh kletok organizma.

It was shown by blot-hybridization with corresponding DNA probes after electrophoretic separation of control and experimental samples of human genome DNA that accumulation of single-strand breaks in the chains of double-strand fragment of transcribing range of ribosomal gene (TRrDNA) does not result in double-strand breaks. That differs from the other studied DNA sequences (cluster of histon genes, Alu-repetition, telomeric repetition and satellite III). Single-strand breaks and double-strand breaks were induced by endonucleases and by gamma-radiation. In spite of higher chemical modification of TRrDNA by arylazide and dimethylsulfate (because of high content of GC-pairs), under the following fragmentation TRrDNA was found to be more resistant to double-strand breaks than other studied DNA sequences. At the same time in the range of non-transcribing spacer (NTS) of ribosomal gene, the section with higher sensitivity to double-strand breaks was found. Higher resistance of TRrDNA to double breaks makes it possible to identify these fragments in cell material from different tissue after death or in DNA samples after prolonged storage. Resistance of TRrDNA to formation of double-strand breaks can be used for its detection in biological fluids after cell death, including the death initiated by ionizing radiation.

[Lymphocyte subpopulation of human peripheral blood responds to the low doses of ionizing radiation,interleukin-2 and to both factors]. / Subpopuliatsiia limfotsitov perifericheskoi krovi cheloveka, ékstre mal'no otvechaiushchaia na vozdeistvie malykh doz ioniziruiushchei radiatsii, interleikina-2 i sovmestnoe vliianie étikh faktorov.

Increasing of 3H-thymidine incorporation in lymphocytes of human peripheral blood which depends non-linearly on X-ray dose (3 cGy max) and interleukin-2 (IL-2) concentration (17.5 Units/ml) is shown. However addition of IL-2 (17.5 U/ml) into the medium of cells after irradiation (3 cGy) decreases almost to the control the effects induced by independently shown actions. Lymphocytes subpopulation responsible for the described phenomena are isolated during the fractionation of lymphocytes in the density gradient and pH (V-fraction BSA). Cell fraction less than 1-2% from the isolated lymphocytes is characterized by increasing of spontaneous corporation of 3H-thymidine, large sizes (d > 8 mkm), decreasing repair after UV-irradiation. It is believed that low dose irradiation and IL-2 activate this cell subpopulation of "last reaction", and higher doses of these factors and this both actions stopping 3H-thymidine incorporation initiate apoptosis. The relation of this sell subpopulation and before proposed ontogenetical reserve cells is discussed.

[Potential relationship between mutation process induced by low doses of ionizing radiation, and positional dynamics of chromosomes in nuclei of eukaryotic cells]. / O vozmozhnoi sviazi mutatsionnogo protsessa, indutsirovannogo malymi dozami ioniziruiushchei radiatsii, i pozitsionnoi dinamikoi khromosom v iadrakh kletok éukariot.

The mutation process has many stages. The information presented in this article suggest that a cell exposed to low LET radiation in the low-dose range (up to 1 cGy) must almost completely repair all spontaneous and radiation-induced DNA lesions. But reparation of DNA double-stranded breaks (DSB), which are the basis of genome instability has peculiarity. We have shown that the mechanisms of action of low doses (which initiate natural antimutagenic reactions of resting cells--an adaptive response) are associated with chromosome loci (centromere) movement in a cell nucleus. We suggest that the movement of chromosome loci in cell nucleus is the fundamental mechanism for repair of DSB and switching of the transcription of gene (it is known that in case of lymphoid cells Ikaros-complexes repressor is colocolizated with centromere loci); in particular, of nucleolar transcription activities because the latter is dependent on centromere arrangement. Because the movement of chromosome loci in both the mitotic cycle and under adapting dose on resting cells is much the same it could be assumed that in latter case the cells also lose their functional characteristic for differentiated resting cells. Under chronic exposure to low doses the functional changes can be the cause of organic changes if adapting dose affects the sufficient part of the cells. The role of cells of evolutional or ontogenetic reserve in mutation process is considered.

[Novel biophysical and biological aspects of mechanisms of effects of low doses of ionizing radiation (low LET) on eukaryotic cells]. / O nekotorykh novykh biofizicheskikh i biologicheskikh aspektakh mekhanizmov pri vozdeistvii malykh i blizkikh k nim doz ioniziruiushchikh izluchenii (nizkikh LPE) na kletki éukariotov.

Novel biophysical and biological aspects of eukaryotic cell response to low doses of low-LET radiation are considered in the present paper. The model of universal nuclear target in eukaryotic cells is proposed. The target is considered as a spherical volume homogeneously filled of DNA with adjacent water layer. Thickness of water layer equals to diffusion length of hydroxil radicals hitting DNA. Two subregions are proposed of the cellular membrane with different ability to respond to low doses of ionizing radiation. An original mechanism of repair of DNA double strand breaks (dsb) is described. This mechanism suggests directional conformational movement of separate locuses of homological chromosomes through narrow tube ("throat") between specialised nuclear regions. The fluxes of nuclear liquid induce a force which is responsible for highly elastic chromosome deformation and chromosome movement resulting in proximity of homological chromosomes in the throat. Close proximity is required for DNA dsb repair. All of DNA dsbs have to be repaired in low doses range owing to high genomic stability of eukaryotic cells. However, it has been hypothesized an existence of a small subpopulation of cells with unstable genome named as cells of evolutionary or/and ontogenetical reserve (ER or/and OR). Genomic instability is induced in these cells by epigenetic program. This program results in DNA damage mainly in chromosomal "hot spots". As a result new genetic variants are originated. The ability of cell population in vivo to eliminate or not eliminate Er/OR cells determinates individual sensitivity of organism and hence health consequences to low dose radiation exposure.

[Biophysical modeling of radiation damage in DNA and chromatin induced by radiation of varying quality]. / Biofizicheskoe modelirovanie radiatsionnykh povrezhdenii DNK i khromatina, indutsirovannykh izlucheniem raznogo kachestva.

DNA double strand breaks in chromatin induced by high LET ionizing radiation are analysed on the basis of biophysical modelling and computer simulation. Theoretical and experimental data are presented arguing in favour of nonrandom clustered distribution of DNA double strand breaks in chromatin.

[Changes induced by low doses of ionizing radiation in the accessibility to DNA restriction enzymes of the ribosomal gene system of human lymphocytes]. / Izmeneniia dostupnosti fermentam restriktsii DNK sistemy ribosomnykh genov limfotsitov cheloveka, indutsirovannye malymi dozami ioniziruiushchego izlucheniia.

Human lymphocytes were X-irradiated at doses 0-10(-2) Gy and allowed to repair for some time. Nuclei were prepared and digested with restriction endonuclease Rsa I to selectively release 28S RNA gene fragment fraction, containing the DNA-binding protein, Hpa II digestion of nuclei was used to investigate the methylation of the 28S RNA gene fragment. There was a differential enrichment of 28S RNA gene binding protein for different X-ray doses with maximum enrichment for dose 2-3 x 10(-2) Gy with following diminish to 10 x 10(-2). The enrichment of less methylated fractions of 28S RNA gene was observed during X-irradiation. This might be explained by a different X-ray-induced changes of methylated and unmethylated rDNA binding with nuclear proteins. The possible mechanism for this phenomena are discussed.