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Gorno-Altaysk, Russia

Orishchenko K.E.,RAS Institute of Cytology and Genetics | Ryzhikova S.L.,CJSC Vector best | Druzhinina Y.G.,CJSC Vector best | Ryabicheva T.G.,CJSC Vector best | And 16 more authors.
Cancer Therapy | Year: 2013

Nucleic acids of various origin and form are capable of activating the innate immune cells and inducing the development of the adaptive immune response. Here we demonstrated that fragments of double-stranded DNA (dsDNA) preparation reached the nuclear space of ex vivo generated human dendritic cells. We showed that dendritic cells generated according to two protocols using IL-4 or IFN-α, upon induction by dsDNA preparation, produced a broad array of cytokines (IFN-γ, MIP-1β, TNF-α, IL-6, G-CSF, and MCP-1). Culturing of human whole blood cells in the presence of dsDNA preparation gave rise to a spectrum of cytokines whose inductive strength considerably surpassed that of pharmacopoeian immunomodulatory preparations (Ridostin, Dezoxyl) and was comparable to that of poly(I):poly(C) preparation or a mixture of mitogens (PHA-P, PHA-M, ConA, and LPS mixture). In a group of relatively healthy donors (n=14), we showed that human dsDNA preparation induced the production of TNF-α, IFN-γ, IL-1RA, IL-1β, IL-6, IL-8, IL-10, G-CSF, and GM-CSF to a great extent and that of IFN-α, VEGF, MCP-1, and IL-18 to a lesser one or not in all the donors by peripheral blood mononuclear cells; human dsDNA preparation was without appreciable effect on the production of IL-2 and IL-17. It was slightly weaker than the standard inducer (the mitogen mixture) with respect to the inductive effect.

Dolgova E.V.,RAS Institute of Cytology and Genetics | Efremov Y.R.,RAS Institute of Cytology and Genetics | Orishchenko K.E.,RAS Institute of Cytology and Genetics | Andrushkevich O.M.,RAS Institute of Cytology and Genetics | And 11 more authors.
Gene | Year: 2013

We previously reported that fragments of exogenous double-stranded DNA can be internalized by mouse bone marrow cells without any transfection. Our present analysis shows that only 2% of bone marrow cells take up the fragments of extracellular exogenous DNA. Of these, ~. 45% of the cells correspond to CD34. + hematopoietic stem cells. Taking into account that CD34. + stem cells constituted 2.5% of the total cell population in the bone marrow samples analyzed, these data indicate that as much as 40% of CD34. + cells readily internalize fragments of extracellular exogenous DNA. This suggests that internalization of fragmented dsDNA is a general feature of poorly differentiated cells, in particular CD34. + bone marrow cells.When linearized plasmid DNA was used as a source of exogenous DNA, we observed that exonucleolytic processing and ligation of double-stranded DNA termini occurred in the bone marrow cells that had this DNA internalized. We also recovered "hybrid" plasmids that encompass kanamycin-resistance gene from the exogenous plasmid DNA and the fragments of plasmids from host enterobacteria, which is suggestive of recombination events taking place upon DNA internalization.CD34. + cells make up the distinctive bone marrow cell population that internalizes extracellular DNA. Cell cycle analysis of CD34. + cells treated with cyclophosphamide only or in combination with dsDNA, suggests that these cells have distinct biologic responses to these treatments. Namely, whereas upon cyclophosphamide treatment bone marrow stem cells become arrested at S-G2 phases, combined cyclophosphamide. +. dsDNA treatment leads to cell cycle progression without any delay. This indicates that when the genome is undergoing repair of interstrand crosslinks, injection of fragmented exogenous dsDNA results in immediate reconstitution of genome integrity. We observe that cyclophosphamide-only or a combined cyclophosphamide. +. dsDNA treatment of cells lead to two distinct waves of apoptosis in CD34. + progenitors. We also show that cyclophosphamide and cyclophosphamide. +. dsDNA injections promote division of CD34. + cells at distinct time periods. © 2013 Elsevier B.V.

Dolgova E.V.,RAS Institute of Cytology and Genetics | Alyamkina E.A.,RAS Institute of Cytology and Genetics | Efremov Y.R.,Novosibirsk State University | Nikolin V.P.,RAS Institute of Cytology and Genetics | And 18 more authors.
Cancer Biology and Therapy | Year: 2014

It has been established previously that up to 40% of mouse CD34+ hematopoietic stem cells are capable of internalizing exogenous dsDNA fragments both in vivo and ex vivo. Importantly, when mice are treated with a combination of cyclophosphamide and dsDNA, the repair of interstrand crosslinks in hematopoietic progenitors is attenuated, and their pluripotency is altered. Here we show for the first time that among various actively proliferating mammalian cell populations there are subpopulations capable of internalizing dsDNA fragments. In the context of cancer, such dsDNA-internalizing cell subpopulations display cancer stem cell-like phenotype. Furthermore, using Krebs-2 ascites cells as a model, we found that upon combined treatment with cyclophosphamide and dsDNA, engrafted material loses its tumor-initiating properties which we attribute to the elimination of tumor-initiating stem cell subpopulation or loss of its tumorigenic potential. © 2014 Landes Bioscience.

Dolgova E.V.,RAS Institute of Cytology and Genetics | Proskurina A.S.,RAS Institute of Cytology and Genetics | Nikolin V.P.,RAS Institute of Cytology and Genetics | Popova N.A.,Novosibirsk State University | And 11 more authors.
Gene | Year: 2012

Morbidity and mortality in mice were observed upon administration of exogenous DNA following their pre-treatment with a cytostatic agent cyclophosphamide. Upon intraperitoneal injections, the fragments of exogenous DNA reached bone marrow cells. These cells were also found to internalize up to 1800. kb of exogenous DNA ex vivo. The 18-24. h time frame represents a final stage in the repair of DNA double-strand breaks, so when exogenous DNA was administered within this critical period of time, pathological changes were observed in many target organs. Namely, bone marrow cells underwent a sustained increase in apoptosis. Copy number of B1 and B2 DNA repeats in bone marrow cells remained unchanged, whereas in the control group of animals their levels were significantly decreased. Finally, the bone marrow cells of moribund animals completely lacked lymphoid progenitors, yet the CD34. + hematopoietic stem cell counts were normal. Histopathology analysis suggested that mice died due to accidental involution of lymphoid organs combined with a systemic inflammatory process induced by massive administration of exogenous DNA and depletion of lymphoid lineage. © 2011 Elsevier B.V.

Alyamkina E.A.,Novosibirsk State University | Nikolin V.P.,RAS Institute of Cytology and Genetics | Popova N.A.,Novosibirsk State University | Dolgova E.V.,Novosibirsk State University | And 10 more authors.
Genetic Vaccines and Therapy | Year: 2010

Background: Immunization of mice with tumor homogenate after combined treatment with cyclophosphamide (CP) and double-stranded DNA (dsDNA) preparation is effective at inhibition of growth of tumor challenged after the treatment. It was assumed that this inhibition might be due to activation of the antigen-presenting cells. The purpose was to develop improved antitumor strategy using mice. We studied the combined action of cytostatics doxorubicin (Dox) plus CP with subsequent dsDNA preparation on tumor growth.Methods: Three-month old CBA/Lac mice were used in the experiments. Mice were injected with CP and human dsDNA preparation. The percentage of mature dendritic cells (DCs) was estimated by staining of mononuclear cells isolated from spleen and bone marrow 3, 6, and 9 days later with monoclonal antibodies CD34, CD80, and CD86. In the next set of experiments, mice were given intramuscularly injections of 1-3 × 105tumor cells. Four days later, they were injected intravenously with 6-6.7 mg/kg Dox and intraperitoneally with 100-200 mg/kg CP; 200 mkg human DNA was injected intraperitoneally after CP administration. Differences in tumor size between groups were analyzed for statistical significance by Student's t-test. The MTT-test was done to determine the cytotoxic index of mouse leucocytes from treated groups.Results: The conducted experiments showed that combined treatment with CP and dsDNA preparation produce an increase in the total amount of mature DCs in vivo. Treatment of tumor bearers with preparation of fragmented dsDNA on the background of pretreatment with Dox plus CP demonstrated a strong suppression of tumor growth in two models. RLS, a weakly immunogenic, resistant to alkalyting cytostatics tumor, grew 3.4-fold slower when compared with the control (p < 0.001). In experiment with Krebs-2 tumor, only 2 of the 10 mice in the Dox+CP+DNA group had a palpable tumor on day 16. The cytotoxic index of leucocytes was 86.5% in the Dox+CP+DNA group, but it was 0% in the Dox+CP group.Conclusions: Thus, the set of experiments we performed showed that exogenous dsDNA, when administered on the background of pretreatment with Dox plus CP, has an antitumor effect possibly due to DC activation. © 2010 Alyamkina et al; licensee BioMed Central Ltd.

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