Publications by authors named "Konstantin E Orishchenko"

18 Publications

  • Page 1 of 1

Recent Advances in the Development of Exogenous dsRNA for the Induction of RNA Interference in Cancer Therapy.

Molecules 2021 Jan 29;26(3). Epub 2021 Jan 29.

Department of Genetic Technologies, Novosibirsk State University, Novosibirsk 630090, Russia.

Selective regulation of gene expression by means of RNA interference has revolutionized molecular biology. This approach is not only used in fundamental studies on the roles of particular genes in the functioning of various organisms, but also possesses practical applications. A variety of methods are being developed based on gene silencing using dsRNA-for protecting agricultural plants from various pathogens, controlling insect reproduction, and therapeutic techniques related to the oncological disease treatment. One of the main problems in this research area is the successful delivery of exogenous dsRNA into cells, as this can be greatly affected by the localization or origin of tumor. This overview is dedicated to describing the latest advances in the development of various transport agents for the delivery of dsRNA fragments for gene silencing, with an emphasis on cancer treatment.
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http://dx.doi.org/10.3390/molecules26030701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865971PMC
January 2021

Natural and Artificial Mechanisms of Mitochondrial Genome Elimination.

Life (Basel) 2021 Jan 20;11(2). Epub 2021 Jan 20.

Federal Research Center Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia.

The generally accepted theory of the genetic drift of mitochondrial alleles during mammalian ontogenesis is based on the presence of a selective bottleneck in the female germline. However, there is a variety of different theories on the pathways of genetic regulation of mitochondrial DNA (mtDNA) dynamics in oogenesis and adult somatic cells. The current review summarizes present knowledge on the natural mechanisms of mitochondrial genome elimination during mammalian development. We also discuss the variety of existing and developing methodologies for artificial manipulation of the mtDNA heteroplasmy level. Understanding of the basics of mtDNA dynamics will shed the light on the pathogenesis and potential therapies of human diseases associated with mitochondrial dysfunction.
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http://dx.doi.org/10.3390/life11020076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909434PMC
January 2021

Functional Evaluation of a Rare Variant c.516G>C (p.Trp172Cys) in the (Connexin 26) Gene Associated with Nonsyndromic Hearing Loss.

Biomolecules 2021 01 5;11(1). Epub 2021 Jan 5.

Federal Research Center Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia.

Mutations in the gene encoding transmembrane protein connexin 26 (Cx26) are the most common cause for hearing loss worldwide. Cx26 plays a crucial role in the ionic and metabolic homeostasis in the inner ear, indispensable for normal hearing process. Different pathogenic mutations in the gene can affect all stages of the Cx26 life cycle and result in nonsyndromic autosomal recessive (DFNB1) or dominant (DFNA3) deafness and syndromes associating hearing loss with skin disorders. This study aims to elucidate the functional consequences of a rare variant c.516G>C (p.Trp172Cys) found with high frequency in deaf patients from indigenous populations of Southern Siberia (Russia). The substitution c.516G>C leads to the replacement of tryptophan at a conserved amino acid position 172 with cysteine (p.Trp172Cys) in the second extracellular loop of Cx26 protein. We analyzed the subcellular localization of mutant Cx26-p.Trp172Cys protein by immunocytochemistry and the hemichannels permeability by dye loading assay. The knockout HeLa cell line has been generated using CRISPR/Cas9 genome editing tool. Subsequently, the HeLa transgenic cell lines stably expressing different variants (wild type and mutations associated with hearing loss) were established based on knockout cells and used for comparative functional analysis. The impaired trafficking of mutant Cx26-p.Trp172Cys protein to the plasma membrane and reduced hemichannels permeability support the pathogenic effect of the c.516G>C (p.Trp172Cys) variant and its association with nonsyndromic hearing loss. Our data contribute to a better understanding of the role of mutations in the second extracellular loop of Cx26 protein in pathogenesis of deafness.
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http://dx.doi.org/10.3390/biom11010061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824951PMC
January 2021

Two Separate Cases: Complex Chromosomal Abnormality Involving Three Chromosomes and Small Supernumerary Marker Chromosome in Patients with Impaired Reproductive Function.

Genes (Basel) 2020 12 17;11(12). Epub 2020 Dec 17.

Institute of Cytology and Genetics, The Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia.

For medical genetic counseling, estimating the chance of a child being born with chromosome abnormality is crucially important. Cytogenetic diagnostics of parents with a balanced karyotype are a special case. Such chromosome rearrangements cannot be detected with comprehensive chromosome screening. In the current paper, we consider chromosome diagnostics in two cases of chromosome rearrangement in patients with balanced karyotype and provide the results of a detailed analysis of complex chromosomal rearrangement (CCR) involving three chromosomes and a small supernumerary marker chromosome (sSMC) in a patient with impaired reproductive function. The application of fluorescent in situ hybridization, microdissection, and multicolor banding allows for describing analyzed karyotypes in detail. In the case of a CCR, such as the one described here, the probability of gamete formation with a karyotype, showing a balance of chromosome regions, is extremely low. Recommendation for the family in genetic counseling should take into account the obtained result. In the case of an sSMC, it is critically important to identify the original chromosome from which the sSMC has been derived, even if the euchromatin material is absent. Finally, we present our view on the optimal strategy of identifying and describing sSMCs, namely the production of a microdissectional DNA probe from the sSMC combined with a consequent reverse painting.
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http://dx.doi.org/10.3390/genes11121511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766715PMC
December 2020

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression.

J Cell Biochem 2019 10 20;120(10):17208-17218. Epub 2019 May 20.

Department of Molecular Mechanisms of Development, Institute of Cytology and Genetics Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type-specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III β-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.
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http://dx.doi.org/10.1002/jcb.28981DOI Listing
October 2019

Nonadherent Spheres With Multiple Myeloma Surface Markers Contain Cells that Contribute to Sphere Formation and Are Capable of Internalizing Extracellular Double-Stranded DNA.

Clin Lymphoma Myeloma Leuk 2016 10 23;16(10):563-576. Epub 2016 Jun 23.

Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Medical Sciences, Novosibirsk, Russia. Electronic address:

Background: The most prominent features of cancer stem cells are asymmetric cell division, tumorigenicity, and clonogenicity. Recently one more feature of poorly differentiated cell types of various origin, including cancer stem cells, has been described. Namely, these cells can internalize extracellular DNA natively, without additional transfection procedures.

Patients And Methods: Using our approach to trace internalization of a TAMRA (carboxy tetramethyl-rhodamine [fluorescent dye])-DNA labeled probe by poorly differentiated cell types, we isolated and characterized the cells from free-floating spheres derived from the bone marrow clonogenic aspirate of a multiple myeloma patient.

Results: Nonadherent spheres display a B-cell phenotype (CD73/CD20/CD45/CD19). Further, free-floating spheres contain 1% to 3% cells with a clonogenic potential, and these cells display a marker of poorly differentiated cell types (TAMRA). Upon association with a group of ∼ 10 free-floating TAMRA cells, this peculiar cell type forms a sphere-forming cluster that initiates secondary aggregation of cells into a spheric structure. TAMRA and TAMRA cells secrete distinct sets of cytokines indicative of the paracrine regulation. Grafting experiments of intact whole spheres versus cell suspensions prepared from dispersed spheres indicate that successful engraftment only occurs in the former case.

Conclusion: Nonadherent 3-D cell colonies (spheres) encompass B cells with CD73/CD20/CD45/CD19 phenotype, as well as double-stranded DNA-internalizing cells. The latter cell type appears to function as a sphere-forming center. Different cells in the spheres communicate with each other by secreting specific sets of cytokines. For successful engraftment and tumor growth in mice, intact spheres containing ∼ 10 cells must be used.
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http://dx.doi.org/10.1016/j.clml.2016.06.014DOI Listing
October 2016

Five-year disease-free survival among stage II-IV breast cancer patients receiving FAC and AC chemotherapy in phase II clinical trials of Panagen.

BMC Cancer 2016 08 18;16:651. Epub 2016 Aug 18.

Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 10 Lavrentieva Ave, Novosibirsk, 630090, Russia.

Background: We report on the results of a phase II clinical trial of Panagen (tablet form of fragmented human DNA preparation) in breast cancer patients (placebo group n = 23, Panagen n = 57). Panagen was administered as an adjuvant leukoprotective agent in FAC and AC chemotherapy regimens. Pre-clinical studies clearly indicate that Panagen acts by activating dendritic cells and induces the development of adaptive anticancer immune response.

Methods: We analyzed 5-year disease-free survival of patients recruited into the trial.

Results: Five-year disease-free survival in the placebo group was 40 % (n = 15), compared with the Panagen arm - 53 % (n = 51). Among stage III patients, disease-free survival was 25 and 52 % for placebo (n = 8) and Panagen (n = 25) groups, respectively. Disease-free survival of patients with IIIB + C stage was as follows: placebo (n = 6)-17 % vs Panagen (n = 18)-50 %.

Conclusions: Disease-free survival rate (17 %) of patients with IIIB + C stage breast cancer receiving standard of care therapy is within the global range. Patients who additionally received Panagen demonstrate a significantly improved disease-free survival rate of 50 %. This confirms anticancer activity of Panagen.

Trial Registration: ClinicalTrials.gov NCT02115984 from 04/07/2014.
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http://dx.doi.org/10.1186/s12885-016-2711-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990870PMC
August 2016

Results of multicenter double-blind placebo-controlled phase II clinical trial of Panagen preparation to evaluate its leukostimulatory activity and formation of the adaptive immune response in patients with stage II-IV breast cancer.

BMC Cancer 2015 Mar 13;15:122. Epub 2015 Mar 13.

LLC Panagen, Gorno-Altaisk, 649000, Russia.

Background: We performed a multicenter, double-blind, placebo-controlled, phase II clinical trial of human dsDNA-based preparation Panagen in a tablet form. In total, 80 female patients with stage II-IV breast cancer were recruited.

Methods: Patients received three consecutive FAC (5-fluorouracil, doxorubicin and cyclophosphamide) or AC (doxorubicin and cyclophosphamide) adjuvant chemotherapies (3 weeks per course) and 6 tablets of 5 mg Panagen or placebo daily (one tablet every 2-3 hours, 30 mg/day) for 18 days during each chemotherapy course. Statistical analysis was performed using Statistica 6.0 software, and non-parametric analyses, namely Wilcoxon-Mann-Whitney and paired Wilcoxon tests. To describe the results, the following parameters were used: number of observations (n), median, interquartile range, and minimum-maximum range.

Results: Panagen displayed pronounced leukostimulatory and leukoprotective effects when combined with chemotherapy. In an ancillary protocol, anticancer effects of a tablet form of Panagen were analyzed. We show that Panagen helps maintain the pre-therapeutic activity level of innate antitumor immunity and induces formation of a peripheral pool of cytotoxic CD8+ perforin + T-cells. Our 3-year follow-up analysis demonstrates that 24% of patients who received Panagen relapsed or died after the therapy, as compared to 45% in the placebo cohort.

Conclusions: The data collected in this trial set Panagen as a multi-faceted "all-in-one" medicine that is capable of simultaneously sustaining hematopoiesis, sparing the innate immune cells from adverse effects of three consecutive rounds of chemotherapy and boosting individual adaptive immunity. Its unique feature is that it is delivered via gastrointestinal tract and acts through the lymphoid system of intestinal mucosa. Taken together, maintenance of the initial levels of innate immunity, development of adaptive cytotoxic immune response and significantly reduced incidence of relapses 3 years after the therapy argue for the anticancer activity of Panagen.

Trial Registration: ClinicalTrials.gov NCT02115984 from 04/07/2014.
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http://dx.doi.org/10.1186/s12885-015-1142-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365563PMC
March 2015

Activity of heat shock genes' promoters in thermally contrasting animal species.

PLoS One 2015 20;10(2):e0115536. Epub 2015 Feb 20.

Engelhardt Institute of Molecular Biology RAS, Vavilov str. 32, Moscow, 119991, Russia.

Heat shock gene promoters represent a highly conserved and universal system for the rapid induction of transcription after various stressful stimuli. We chose pairs of mammalian and insect species that significantly differ in their thermoresistance and constitutive levels of Hsp70 to compare hsp promoter strength under normal conditions and after heat shock (HS). The first pair includes the HSPA1 gene promoter of camel (Camelus dromedarius) and humans. It was demonstrated that the camel HSPA1A and HSPA1L promoters function normally in vitro in human cell cultures and exceed the strength of orthologous human promoters under basal conditions. We used the same in vitro assay for Drosophila melanogaster Schneider-2 (S2) cells to compare the activity of the hsp70 and hsp83 promoters of the second species pair represented by Diptera, i.e., Stratiomys singularior and D. melanogaster, which dramatically differ in thermoresistance and the pattern of Hsp70 accumulation. Promoter strength was also monitored in vivo in D. melanogaster strains transformed with constructs containing the S. singularior hsp70 ORF driven either by its own promoter or an orthologous promoter from the D. melanogaster hsp70Aa gene. Analysis revealed low S. singularior hsp70 promoter activity in vitro and in vivo under basal conditions and after HS in comparison with the endogenous promoter in D. melanogaster cells, which correlates with the absence of canonical GAGA elements in the promoters of the former species. Indeed, the insertion of GAGA elements into the S. singularior hsp70 regulatory region resulted in a dramatic increase in promoter activity in vitro but only modestly enhanced the promoter strength in the larvae of the transformed strains. In contrast with hsp70 promoters, hsp83 promoters from both of the studied Diptera species demonstrated high conservation and universality.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115536PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336284PMC
January 2016

Delivery and processing of exogenous double-stranded DNA in mouse CD34+ hematopoietic progenitor cells and their cell cycle changes upon combined treatment with cyclophosphamide and double-stranded DNA.

Gene 2013 Oct 30;528(2):74-83. Epub 2013 Jul 30.

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk 630090, Russia.

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.
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http://dx.doi.org/10.1016/j.gene.2013.06.058DOI Listing
October 2013

A regulatory potential of the Xist gene promoter in vole M. rossiaemeridionalis.

PLoS One 2012 11;7(5):e33994. Epub 2012 May 11.

Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Branch, Novosibirsk, Russia.

X chromosome inactivation takes place in the early development of female mammals and depends on the Xist gene expression. The mechanisms of Xist expression regulation have not been well understood so far. In this work, we compared Xist promoter region of vole Microtus rossiaemeridionalis and other mammalian species. We observed three conserved regions which were characterized by computational analysis, DNaseI in vitro footprinting, and reporter construct assay. Regulatory factors potentially involved in Xist activation and repression in voles were determined. The role of CpG methylation in vole Xist expression regulation was established. A CTCF binding site was found in the 5' flanking region of the Xist promoter on the active X chromosome in both males and females. We suggest that CTCF acts as an insulator which defines an inactive Xist domain on the active X chromosome in voles.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033994PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3350511PMC
October 2012

Effects of human exogenous DNA on production of perforin-containing CD8+ cytotoxic lymphocytes in laboratory setting and clinical practice.

Cell Immunol 2012 Mar-Apr;276(1-2):59-66. Epub 2012 Apr 7.

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia.

We investigated the influence of Panagen DNA preparations on laboratory animals and IFN-induced human dendritic cells, as well as analyzed the data from a phase II clinical trial in the therapy of breast cancer. It was shown that this treatment resulted in increased number of CD8+/perforin+ T cells in peripheral lymphoid organs of experimental animals, in mixed lymphocyte culture population and in peripheral blood of breast cancer patients. Moreover, we demonstrated that when Panagen DNA preparations are used in combination with the standard FAC-based breast cancer therapies, non-specific immune response activity remains at the same levels as observed prior to therapy, whereas in FAC-placebo patients, non-specific immunity is greatly diminished.
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http://dx.doi.org/10.1016/j.cellimm.2012.04.004DOI Listing
October 2012

"Delayed death" phenomenon: a synergistic action of cyclophosphamide and exogenous DNA.

Gene 2012 Mar 27;495(2):134-45. Epub 2011 Dec 27.

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk 630090, Russia.

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.
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http://dx.doi.org/10.1016/j.gene.2011.12.032DOI Listing
March 2012

A strategy of tumor treatment in mice with doxorubicin-cyclophosphamide combination based on dendritic cell activation by human double-stranded DNA preparation.

Genet Vaccines Ther 2010 Nov 1;8. Epub 2010 Nov 1.

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia.

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 × 105 tumor 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.
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http://dx.doi.org/10.1186/1479-0556-8-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2987767PMC
November 2010

Effect of double-stranded DNA on maturation of dendritic cells in vitro.

Cell Immunol 2010 21;266(1):46-51. Epub 2010 Sep 21.

Novosibirsk State University, Russia.

A preparation of human genomic fragmented double-stranded DNA (dsDNA) was used as maturation stimulus in cultures of human dendritic cells (DCs) generated in compliance with the interferon protocol. Culturing of the DCs in medium with 5μg/ml of the DNA preparation was associated with a decrease in the relative proportion of CD14 + cells and an increase in that of CD83 + cells. These changes are markers of DC maturation. The efficiency with which the DNA preparation was able to elicit DC maturation was commensurate with that of lypopolysaccharide from bacterial cell, the standard inducer of DC maturation. Generated ex vivo, matured in the presence of the human DNA preparation, pulsed with tumor antigens mouse DCs were used as a vaccine in biological tests for its antitumor activity. The experimental results demonstrate that reinfusion of mature pulsed with tumor antigens DCs cause a statistically significant suppression of tumor graft growth.
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http://dx.doi.org/10.1016/j.cellimm.2010.08.011DOI Listing
December 2010

Exogenous allogenic fragmented double-stranded DNA is internalized into human dendritic cells and enhances their allostimulatory activity.

Cell Immunol 2010 1;262(2):120-6. Epub 2010 Feb 1.

Novosibirsk State University, Novosibirsk, Russia.

Exogenous allogenic DNA as nucleosome-free fragments reaches main cellular compartments (cytoplasm, nucleus) of human dendritic cells and deposits in the nuclear interchromosomal space without visibly changing in linear size. The presence of such allogenic fragmented DNA in medium in which human dendritic cells are cultured produces an enhancement of their allostimulatory activity. This enhancement is comparable to that produced by the standard maturation stimulus lipopolysaccharide Escherichia coli.
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http://dx.doi.org/10.1016/j.cellimm.2010.01.005DOI Listing
May 2010

Combined therapy with cyclophosphamide and DNA preparation inhibits the tumor growth in mice.

Genet Vaccines Ther 2009 Aug 14;7:12. Epub 2009 Aug 14.

Novosibirsk State University, Novosibirsk, Russia.

Background: When cyclophosphamide and preparations of fragmented exogenous genomic double stranded DNA were administered in sequence, the regressive effect on the tumor was synergic: this combined treatment had a more pronounced effect than cyclophosphamide alone. Our further studies demonstrated that exogenous DNA stimulated the maturation and specific activities of dendritic cells. This suggests that cyclophosphamide, combined with DNA, leads to an immune response to the tumors that were grafted into the subjects post treatment.

Methods: Three-month old CBA/Lac mice were used in the experiments. The mice were injected with cyclosphamide (200 mkg per 1 kg body weight) and genomic DNA (of human, mouse or salmon sperm origin). The DNA was administered intraperitoneally or subcutaneously. After 23 to 60 days, one million tumor cells were intramuscularly grafted into the mice. In the final experiment, the mice were pre-immunized by subcutaneous injections of 20 million repeatedly thawed and frozen tumor cells. Changes in tumor growth were determined by multiplying the three perpendicular diameters (measured by caliper). Students' t-tests were used to determine the difference between tumor growth and average survival rate between the mouse groups and the controls.

Results: An analysis of varying treatments with cyclophosphamide and exogenous DNA, followed by tumor grafting, provided evidence that this combined treatment had an immunizing effect. This inhibitory effect in mice was analyzed in an experiment with the classical immunization of a tumor homogenate. The strongest inhibitory action on a transplanted graft was created through the following steps: cyclophosphamide at 200 mg/kg of body weight administered as a pretreatment; 6 mg fragmented exogenous DNA administered over the course of 3 days; tumor homogenate grafted 10 days following the final DNA injection.

Conclusion: Fragmented exogenous DNA injected with cyclophosphamide inhibits the growth of tumors that are grafted to mice after this combined treatment.
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http://dx.doi.org/10.1186/1479-0556-7-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732619PMC
August 2009