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

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

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

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

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

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

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

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

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

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

[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.

[Stress signaling between human lymphocytes after induction of bystander effect by exposure to ionizing radiation in adaptive doses].

We previously reported that the consequence of human lymphocytes irradiation by the adaptive doses (X-rays, 10 cGy) was a transposition of the homologous chromosome loci in the cell nucleus (FISH method); this phenomenon was mediated by the increase of nucleolus activity. They both are transmited to non-irradiated cells by the bystander effect (BE). We shown that the reaction of stress signaling is induced by the DNA fragments of irradiated lymphocytes. The study shows that after the inhibition of caspase 3 activity in irradiating lymphocytes or the blockade TLR9 in bystander cells the transposition was not observed. A signaling way of BE from irradiated lymphocytes apoptosis to bystander cells receptors is discussing.

Low-Dose Ionizing Radiation Affects Mesenchymal Stem Cells via Extracellular Oxidized Cell-Free DNA: A Possible Mediator of Bystander Effect and Adaptive Response.

We have hypothesized that the adaptive response to low doses of ionizing radiation (IR) is mediated by oxidized cell-free DNA (cfDNA) fragments. Here, we summarize our experimental evidence for this model. Studies involving measurements of ROS, expression of the NOX (superoxide radical production), induction of apoptosis and DNA double-strand breaks, antiapoptotic gene expression and cell cycle inhibition confirm this hypothesis. We have demonstrated that treatment of mesenchymal stem cells (MSCs) with low doses of IR (10 cGy) leads to cell death of part of cell population and release of oxidized cfDNA. cfDNA has the ability to penetrate into the cytoplasm of other cells. Oxidized cfDNA, like low doses of IR, induces oxidative stress, ROS production, ROS-induced oxidative modifications of nuclear DNA, DNA breaks, arrest of the cell cycle, activation of DNA reparation and antioxidant response, and inhibition of apoptosis. The MSCs pretreated with low dose of irradiation or oxidized cfDNA were equally effective in induction of adaptive response to challenge further dose of radiation. Our studies suggest that oxidized cfDNA is a signaling molecule in the stress signaling that mediates radiation-induced bystander effects and that it is an important component of the development of radioadaptive responses to low doses of IR.

[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.

[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.

[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.

[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 characteristics of unscheduled DNA synthesis and of the changes in the structural parameters of human lymphocyte nuclei after the action of x-ray radiation in low doses and in combination with UV irradiation]. / Osobennosti vneplanovogo sinteza DNK i izmenenii strukturnykh parametrov iader limfotsitov cheloveka posle deistviia rentgenovskogo izlucheniia v malykh dozakh i v sochetanii s UF-oblucheniem.

Effects of X-ray low doses (0.5-25 cGy) with following UV light (254 nm, 20 J/m2) on human peripheral blood lymphocytes were studied. Reparation response registered by unscheduled DNA synthesis (UDS) activity was demonstrated to be the most intensive after action of X-rays in dose ranges 2-3 cGy and 15-20 cGy, and least intensive after 10 cGy. In those cells where UV light was followed by X-rays, dose ranges 2-3 cGy and 15-20 cGy cause essential decrease of UDS as compared with UV-light action only. The most intensive UDS was in those UV-irradiated lymphocytes which where previously exposed to 10 cGy of X-rays. At the same time 10 cGy cause minimal reparation response without UV light. Possible mechanisms of discovered phenomena are discussed. In particular, reaction on 2-3 cGy might be a reorganization of genome for adaptive response or an evolution reserve cells response. From 10 cGy the reason of reaction might be a reparation, induced by radiation.

[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).

[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.

[The dependence of DNA repair induced by genetically hazardous exposures on the ionic strength of the medium containing the cells]. / Zavisimost' reparatsii DNK, indutsirovannoi geneticheski opasnymi vozdeistviiami, ot ionnoi sily sredy, v kotoroi nakhodiatsia kletki.

The dependence of UV-induced unscheduled DNA synthesis (UDS) in non-stimulated lymphocytes of human peripheral blood on the ionic strength (mu) of the culture medium has been shown. With the level of mu lower or higher than physiological (mu ph) the UDS significantly decreases. The effect of modification of mu due to changes in ionic strength is absent in the lymphocytes of patients with the classic form of xeroderma pigmentosum. This phenomenon may become useful for development of a new test revealing cells with genetically or physiologically changed system of UV-induced DNA repair. Mechanisms of investigated phenomenon, particularly their dependence on the chromatin structure, as well as the influence of ionic strength on binding the repair enzymes with DNA are discussed.

[The effect of the environment on the repair of DNA damage in human lymphocytes]. / Vliianie okruzhaiushchei sredy na reparatsiiu povrezhdenii DNK limfotsitov cheloveka.

The dependence of UV-induced unscheduled DNA synthesis (UDS) in the unstimulated lymphocytes of human peripheral blood on the ionic strength (mu) of the culture medium has been shown. In the level of mu lower or higher than the physiological (mu ph) one, UDS significantly decreases. The effect of modification of mu due to the changes of ionic strength is absent in the lymphocytes of the classic form of xeroderma pigmentosum. The phenomenon may become useful in the development of a new test for revealing cells with a genetically or physiologically changed system of UV-induced DNA repair. The mechanisms of investigated phenomenon, particularly their dependence on the structure of chromatin, as well as the influence of ionic strength on binding of the repair enzymes with DNA are discussed.

[Cell-free DNA fragments increase transcription in human mesenchymal stem cells, activate TLR-dependent signal pathway and supress apoptosis].

Human mesenchymal stem cells (MSCs) are now widely adopted in regenerative medicine. However, many questions on the role of different signaling pathways in the regulation of stem cell (SC) functional activity within the organism remain unaswered. In damaged regions the level of cell death increases and DNA fragments from dead cells (cell-free DNA, cfDNA) are accumulated in blood. We showed that in adipose-derived MSCs exposed in vitro to cfDNA fragments the transcription level increased (the total amount of cellular RNA and the rRNA amount rose). GC-rich CfDNA fragments (GC-DNA) activated the TLR9-dependent signal pathway: the expression of TLR9 and of TLR9-signaling pathway adapter--MyD88--was up-regulated. AT-rich DNA fragments did not increase the TLR9 expression, though, the MyD88 expression level rose. So we suggest that AT-DNA acts via some other receptors that nevertheless activate MyD88-dependent signalling in MSCs. We also showed that cfDNA fragments decreased the activity of caspase, an apoptotic enzyme. So, ctDNA can significantly influence the functional activity ofMSC by activating TLR9- and MyD88-dependent signal pathways and lowering the apoptosis level.