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

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

[Use of the most recent reagent (CuFL) for stimulation of NO synthesis by the medicinal leech salivary cell secretion in the cultures of human endothelium cells (HUVEC) and in rat cardiomiocytes].

The medicinal leech salivary cell secretion (SCS) may stimulate NO-production in cultures of human endothelium cells (HUVEC) and rat cardiomiocytes (RCM). This effect was detected using a NO specific reagent, - the complex Cu2+ with a fluorescein derivative (Cu-Fl). NO had also been detected in the cells by fluorescent electronic microscopy and determined quantitatively in the cells and in culture fluid by the fluorescence method. SCS stimulated NO synthesis in HUVEC cells (but not in RCM) is accompanied by NO release into intercellular space. Localization of NO synthesis centers is presented and it is shown that the increase in NO levels during the SCS action on HUVEC and RCM is associated with the increase in the activity of eNOS/nNOS, but not iNOS. In endothelial cells SCS activates nitrosylation processes, assessed by the increase of nitrite-ions in the culture medium. It is therefore important to use Cu-Fl, other than Griss-reagent, during the first hour of analysis of NO synthesis. The NO-depended mechanism of SCS action on endothelial cells might be a factor in providing of its positive action in hirudotheraphy.

[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.

[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.