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.

Investigation of Thionins from Black Cumin (Nigella sativa L.) Seeds Showing Cytotoxic, Regulatory and Antifungal Activity.

Thionins (NsW1 and NsW2), earlier isolated from the seeds of endemic Middle-Asian black cumin (Aligella sativa L.), showing signilicant inhibitory action on some bacterial and yeast pathogens were investigated for cytotoxic properties against several tumor cell lines (AsPC-1, Colo357, RD and Jukart) in vitro within nano- and micromolar ranges of the active concentrations and as modulators of expression of the genes controlling conversion of normal cells to malignant ones. Suppression of the expression of the genes from MMP, RhoA, miR21 families in human rhabdomyosarcoma (RD) cells was observed, whereas the influence of the molecules on the genes in normal blood cells was not identified. It was shown that the thionins from black cumin induced almost 90% of the cell death in RD and Jukart lines. Moreover, the polypeptides inhibited clinical isolates of Aspergillus ochraceus and A.fimigatus at the level comparable with that of amphotericin B. The data demonstrated that the peptides could be considered as perspective antitumor and antimycotic agents.

[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 peculiarities of polymorphism of XPD and XRCC1 repair genes in cells of Down and Ehlers-Danlo syndrome patients characterized by increased radiosensitivity].

Codon 312 and 751 polymorphisms ofXPD gene and codon 399 polymorphism of XRCC1 gene of peripheral blood lymphocytes in patients with Down syndrome (DS) (46 individuals), Ehlers-Danlo syndrome (EDS) (47 individuals) and in a group of healthy donors (control) (40 individuals) have been studied. Frequency of XPD genotype (G312G) coding for the most effectively functioning form of XPD protein was lower in patients with DS (26%) than in a group of healthy donors (42.5%) (p = 0.035), whereas no significant differences with the control were revealed for this codon in patients with EDS. No patients with XPD genotype (C751C) (p = 0.036) were revealed in a group of EDS patients, while this genotype was found in 16% of a group of healthy donors and in 17% of patients with DS. The trend of XRCC1 genotype frequency reduction (A399A) (p = 0.085) in EDS patients (3.9%) compared with the group of healthy donors (13.5%) and DS patients (13.3%) has been obtained. These data show that polymorphisms of the excision repair genes under study are accompanied by an elevated individual radio sensitivity in patients with DS. Genes investigated (their polymorphic variants) did not participate in the mechanisms for radio sensitive phenotype formation in EDS patients.

[Research of mutations in oncogenes and tumour suppressor genes as the approach to research of ways of the individual prognosis of delayed effects of exposure to ionizing radiation].

The frequency of mutant forms p53 and N-Ras genes was investigated in DNA from peripheral blood of the patients by method Polymerase Chain Reaction - Single Strand Conformation Polymorphism Analysis. The patients were investigated in late period after radiation accidents exhibited acute radiation syndrome. It is established that the mutations among patients in areas of codons 246-250 exon 7 of p53 gene and codon 12 of N-Ras gene were meet more often than in control group. It is shown that these mutations possibly arise in insignificant number of the cells with the radiation-induced genomic instability. Possibility of use of mutations in protooncogenes and tumour suppressor genes as markers of risk of development of the main thing from delayed effects of exposure to ionizing radiation - malignant tumours is discussed.

Effect of oxidized fibrinogen on aggregation of activated platelets and neutrophils.

The effect of oxidized fibrinogen on platelet-neutrophil complex formation was evaluated by studying the platelet aggregation (changes in light transmission and turbidimetric assay). Activation of cells by thrombin (0.015 U/ml) in the presence of oxidized fibrinogen was accompanied by the formation of larger intermolecular aggregates of platelets and leukocytes as compared to those detected in experiments with non-oxidized fibrinogen. Addition of thrombin (0.2 U/ml) in the presence of oxidized fibrinogen was followed by the formation of more stable complexes of platelets and leukocytes as compared to those revealed in experiments with non-oxidized fibrinogen. An increase in the width of aggregation curves was most pronounced in the system of 10(-4) M Fe(2+) and 10(-4) M H(2)O(2) with oxidized fibrinogen. Our results indicate that oxidized fibrinogen contributes to the "floating" or suspension of platelet-leukocyte complexes.

Evaluation of the resistance of blood plasma to oxidative stress by oxidizability of proteins and lipids during metal-catalyzed oxidation.

A new approach for the evaluation of oxidizability of proteins and lipids in the same sample of blood plasma was proposed. We tested a method for evaluation of metal-catalyzed oxidation of fibrinogen by the formation of bityrosine cross-links during oxidation detected by the increase in fluorescence at 415 nm. A correlation was revealed between parameters of oxidizability estimated by this marker and carbonyl derivatives (dinitrophenylhydrazine method). Oxidizability of total proteins from whole plasma was compared with oxidizability of plasma lipids (marker malonic dialdehyde). Study of these parameters in patients with coronary heart disease showed that the proposed experimental approach allows us to divide the sample into several subgroups differing in the resistance to oxidative stress. These data can be used for diagnostic and prognostic purposes.