Changes in the Activity of Genes Involved in the Regulation of Hematopoiesis during Tumorigenesis in Irradiated Mice.

We used 183 F1 CBA×C57Bl hybrid mice to study the delayed effects of low-power long-term γ-irradiation at a dose of 12.6 Gy (10 mGy/min) 8 and 10 months after the treatment. Eight months after the treatment we found the increased expression of the transcription factor NFκB and its target genes iNOS and G-SCF in the bone marrow (BM). Ten months after the treatment malignant lymphomas were revealed in 14 of 94 mice in the liver, abdominal cavity, and subcutaneously. In the BM of these mice, the transcription of the PTEN, NFκB, and iNOS genes was inhibited and the contents of long non-coding RNAs (lncRNAs) lnc p21, NEAT1, and microRNA miR-125b were decreased. The expression of the NFκB(p65) gene and miR-125b was inhibited in the BM of irradiated mice without tumors ten months after the treatment. These data show the deregulation of the P53 system supporting the genome stability in the BM of irradiated mice. These indices will be studied as potential markers of risk of development of irradiation-induced tumors.

[Searching Radiation Countermeasures using the Model of Prolonged Irradiation of Mice with Low Dose Rate and Evaluation of Their Influence on Heat Shock Protein Genes Expression].

Different radiomodificators (cytokine betaleukine, antioxidant phenoxan, antigipoksant limontar and nucleoside riboxin) were investigated on mice for evaluating their radiation protective capacity against prolonged (21 h) exposure at a dose of 12.6 Gy at a low dose rate of 10 mGy/min. Bone marrow cellularity and endogenic CFUs were used as evaluation criteria 9 days after exposure. Simultaneously, expression of the heat shock proteins of 25, 70 and 90 kDa in unexposed mice bone marrow was studied 2, 24 and 48 h after injections. Betaleukine only had a positive significant effect in both tests in the variants of 50 mcg/kg and 3 mcg/kg when administered 2 h and 22 h before exposure, correspondingly. Effects of betaleukine HSPs on expression were both stimulating and inhibiting, that was in contradiction with a constant positive effect in 5 experiments on exposed mice for each betaleukine variant. It argues against the vital role of HSPs in the betaleukine antiradiation effect. In 2 experiments with high temperatures betaleukine administered at a dose of 50 mcg/kg evoked a very high HSP-70 gene expression after 24 h, and mice exposed to irradiation at that time in a parallel experiment showed an increased radiation effect. It corresponds to the idea that HSPs serve a stress indicator.

[Comparative analysis of natural and synthetic antimutagens as regulators of gene expression in human cells under exposure to ionizing radiation].

This paper studies the effect of plant peptides of thionine Ns-W2 extracted from seeds of fennel flower (Nigella sativa) and ß-purothionine from wheat germs (Triticum kiharae), as well as a synthetic antimutagen (crown-compound), on the expression of several genes involved in the.control of cellular homeostasis, processes of carcinogenesis, and radiation response in human rhabdomyosarcoma cells (RD cells), T-lymphoblastoid cell line Jurkat, and blood cells. All of these agents acted as antimutagens-anticarcinogens, reducing the expression of genes involved in carcinogenesis (genes of families MMP, TIMP, and IAP and G-protein genes) in a tumor cell. A pronounced reduction in the mRNA level of these genes was caused by thionine Ns-W2, and the least effect was demonstrated by ß-purothionine. Antimutagens had very little effect on the mRNA levels of the several studied genes in normal blood cells.

[Expression of P53, NPM1, Kras, c-Myc, p14(ARF) genes in blood cells of cancer patients before and after radiation therapy].

The mRNA levels of P53gene, as well as NPM1, Kras, c-Myc, p14(ARF) genes, which, according to the published data, code for the proteins regulating the p53 activity, were studied using RT-PCR method in blood cells of patients with different localization of tumor process (prostate cancer, breast cancer and head and neck cancer) before and after application of radiation therapy. Changes in gene expression of cancer patients were compared with the control group of healthy donors. We have established that all patients had a decreased level of the Kras gene expression even before radiotherapy; moreover, the group of patients with prostate cancer had a low content of mRNA in NPM1 and p14(ARF), and the group of patients with head and neck cancerhad a reliably reduced mRNA in P53, NPM1 and p14(ARF). The radiation therapy did not cause essential changes in the expression of these genes of cancer patients, ecpect for the Kras gene, whose the mRNA level in the group of patients with head and neck cancer was reliably lower than the mRNA level prior to beginning of radiation therapy. The correlations of P53, NPM1, Kras, p14(ARF) gene expression were studied. We have shown that p14(ARF) mRNA level negatively correlates with Kras mRNA (R = -0.6, p = 0.002) and P53 mRNA levels (R = -0.49, p = 0.013) in the control group of healthy donors. A positive correlation was observed between P53 mRNA and NPM1 mRNA (R = 0.54, p = 0.006). Similar correlations between mRNA levels of these genes in blood cells were absent in the cancer patients before radiotherapy. After radiotherapy in patients with prostate cancer, p14(ARF) mRNA level positively correlated with NPM1 mRNA (R = 0.7, p = 0.001) and negatively with Kras mRNA (R = - 0.5, p = 0.03). Our results provide evidence that expression P53, NPM1, Kras and p14(ARF) genes may be coordinated in blood cells of healthy donors. The low expression levels of the studied genes in patients can contribute to the increase in the mutation changes in blood cells of the examined subjects after the action of genotoxic factors.

[The comparison of molecular-biochemical and cytogenetic analyses of blood cells of patients long after the acute radiation sickness].

Molecular-biochemical and cytogenetic analyses were made on blood cells of 17 radiation accident victims who, from 1.7 to 43.8 years previously, had suffered acute radiation sickness (ARS) ranging from severity grades I to IV. Molecular-biochemical data were obtained with patients' leukocytes and with mononuclear cells on their oxidative status by a) the level of an anion-radical O2*- in the 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2-tetrazolium bromide (MTT) test and b) the sum of reactive oxygen species in the 2,7-dichlorodihydrofluorescein diacetate (DCFDA) test together with a test for DNA strand unwinding in alkaline conditions by measurements of fluorescence intensity of ethidium bromide. Each parameter was measured in freshly sampled cells and during a 5-hour incubation as absolute means on 5 measurements and in % change from the initial values. Cytogenetic data were obtained from the standard metaphase preparations scored for routine unstable chromosomal aberrations (us-CA)--dicentrics; and stable aberrations (sCA)--translocations. The latter ones were assayed by the FISH method using whole chromosome 2, 4 and 12 fluorescent probes and scaled up to genome equivalence. For all patients reduced oxidative status of about 25-30% was obtained by the MTT-test (p < 0.005), and the DCFDA-test (p < 0.027). The yield of usCA depended on the time after irradiation with higher yields associated with the shorter postirradiation times and reducing almost to expected background frequencies. The yield of sCA was high for all patients, correlating with the severity of ARS whilst the molecular-biochemical parameters showed no relationship with ARS. A correlation was observed between parameters of oxidative status and % of cells with usCA: by the MTT-test r = from 0.50 up to 0.61 (p = from 0.06 up to 0.003), but by the DCFDA-test the strength of correlation was smaller: r = from 0.38 up to 0.48. An inverse correlation was found between initial oxidative state of mononuclear cells and the frequency of CA in lymphocytes. Similarly a marked inverse correlation between degree of DNA unwinding by the ethidium bromide assay on leukocytes and sCA in lymphocytes was also noted. The feasibility of radiation-induced delayed genomic instability in vivo for humans long time after irradiation is discussed.

[Molecular manifestations of radiation-induced genome instability: the possibility of chemical modification].

The molecular manifestations of radiation-induced genome instability-changes of the DNA structure, the excision DNA repair and the contents of the reactive oxygen forms in bone marrow cells of the repair proficient mice (CBA) and of the repair-defective (101/H) lines in the dynamics up to 185 day after ionizing radiation exposure in the dose of 1.5 Gy were studied. Is was established, that after irradiation in bone marrow cells the descendants with the decreased activity of excision DNA repair and prone to increased changes of DNA structure DHK is arised. The injection of the phenozane in concentrations causing its receptor interaction with cells, did not defend DNA of the bone marrow cells from the radiation injury after the exposure in a sublethal dose, however it exerted influence on long-term changes. Due to the phenosane of the bone marrow cells of the irradiated mice of CBA line exhibited the larger activity in a DNA repair from damages and maintenance of vitality. The bone marrow cells of male mice of repair defective 101/H line, which phenozan was entered before the irradiation, remained unfit to the remuval of DNA damages by the repair, that probably resulted the activations of the program of the maintenance of genome constancy by the apoptosis in the cells--carriers of the structural defects and the cause of animal lethality.

[Sexual differences in a state systems of DNA structure maintenance and generation of reactive oxygen species in somatic cells of mice 101/H reparation-defective strain and manifestation of these differences after exposure to ionizing radiation]. / Polovye razlichiia sostoianiia sistem podderzhaniia struktury DNK i generatsii aktivnykh form kisloroda v somaticheskikh v somaticheskikh kletkakh u myshei repearatsionno-defektivnoi 101/H i ikh proiavleniia pri vozdeistvii ioniziruiushchei radiatsii.

To analyse a role of the factor of a genetic fundamentals of cells in formation of radiation-induced genome instability (RIGI) we investigated a condition of DNA pattern, content of superoxide anion-radical O2*- and a sum of reactive oxigen species (ROS) (O2*-, OH*, H2O2), and also catalase activity in bone marrow cells of male and female mice of 101/H strain in the norm and at once after chronic (10 day) exposure to 200 mGy gamma-radiation. Thus we based on conception about a significance of mechanisms of DNA repair and production of reactive oxygen species (ROS) in development of radiation-induced genome instability (RIGI), and also on the data on sex bound differences in efficiency of DNA repair in reply to impact of the genotoxic agents for male and female mice of 101/H strain. Sex connected differences in redox system of bone marrow cells were established. In males lower catalase activity was found in the norm, with considerable increase of the activity and the content of ROS after chronic irradiation with a low dose (200 mGy); at the same time a direct correlation between the ROS content and catalase activity occurred. In female, which have higher DNA repair potential, higher level of catalase activity was found in the norm, with reduction after irradiation and lower, than in male, level of O2*- content; no changes in the general ROS content, or direct correlation between the content of a superoxide anion-radical (O2*-) and the sum of ROS were observed. The detected differences between male and female the studied parameters in the norm and after irradiation indicate a connection of the studied characteristics and their changes with a sex, confirm the literature data about a significance of the factor of a genetic fundamentals of bioobject in formation of radiation-induced genome instability.

[Interrelations between the content of reactive oxygen species and the state of DNA structure in bone marrow cells of mice after whole body gamma-irradiation]. / Vzaimosviaz' soderzhaniia aktivnykh form kisloroda i sostoianiia struktury DNK v kletkakh kostnogo mozga u myshei v dinamike posle obshchego vozdeistviia gamma-izlucheniia.

The aim of the research is a further analysis of a problem concerning two (regulatory and damaging) functions of reactive oxygen species (ROS) in viability of organism cells under acute exposure to ionizing radiation. For this purpose the ROS content and the state of DNA structure in bone marrow cells of male CBA and SHK mice have been studied in dynamics, from 15 minutes up to 185 day after acute exposure to a sublethal dose (1.5 Gy) of ionizing radiation. The analysis of dependencies between these parameters in the norm, immediately after irradiation and in later cell descendants showed the direct correlation between the ROS content and the DNA nativity in the norm; 185 days after irradiation the correlation disappeared. It was suggested that the correlation occurred in the norm indicates participation of the ROS (as a sensory link) in a system of reactions (under the control of the corresponding genetic program), that ensure the DNA structure and, ultimately, the genome stability. The loss of such connection after acute exposure to ionizing radiation indicates actuation of another module of reactions sustaining stability of cellular genome in new conditions, without regulatory participation of ROS, that can promote or demonstrate the development of radiation-induced genome instability.

[Indometofen causes biochemical changes in the blood cells characteristic for a radioresistant state of the body]. / Indometofen vyzyvaet v kletkakh krovi biokhimicheskie izmeneniia, kharakternye dlia radiorezistentnogo sostoianiia organizma.

The DNA and RNA contents, RNA/DNA ratio, and spontaneous and latex-induced oxidant activity indices of the whole blood were studied in the nitroblue tetrazolium test of mono- and polymorphonuclear blood leucocytes of intact dogs after injection of lipopolysaccharide pyrogenal. Significant changes in the above parameters were revealed for radioresistant (survived) and radiosensitive (lost) animals exposed to a subsequent prolonged gamma irradiation with a lethal dose of 7.64 Gy (LD75/45). Peroral introduction of 30 mg/kg indometofen (an indole analog of tamoxifen), which is a potential radioprotector, to dogs increased the survival rates of the irradiated dogs up to 93% and aided in the adaptive biochemical changes in the nuclear cell compartment of blood to induce a radioresistant status of the organism.

[The effect of the potential antiradiation agent compound HC-1539 on reparative DNA synthesis induced by the damaging action of the chemical mutagen N-nitroso-N-methylurea and radiation]. / Vliianie potentsial'nogo protivoluchevogo sredstva--soedineniia HC-1539--na reparativnyi sintez DNK, indutsiruemyi povrezhdaiushchim deistviem khimicheskogo mutagena N-nitrozo-N-metilmocheviny i izluchenii.

The influence of NS-1539, a potential anti-irradiation agent and indole analogues of tamoxifen on the DNA repair synthesis in the bone marrow and lymphocytes of peripheral blood was studied. It was found that the DNA repair processes from damages caused by chemical mutagen N-nitroso-N-methylurea or physical factor UV-irradiation in the bone marrow cells after the NS-1539 protection of mice as well as in lymphocytes of peripheral blood of human donors were enhanced. The enhancing of DNA repair synthesis was seen not earlier than 18 hours after the NS-1539 injection to mice in vivo or when NS-1539 was added to lymphocyte suspension in vitro and observed up to the third day. This period of time coincides with the time of formation of radioresistance of organism under the influence of this agent as well as with the time of realization effects of steroid hormones on target cells.