Konstantin E. Orishchenko

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

BackgroundImmunization 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.MethodsThree-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 Students t-test. The MTT-test was done to determine the cytotoxic index of mouse leucocytes from treated groups.ResultsThe 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.ConclusionsThus, 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.

“Delayed death” phenomenon: A synergistic action of cyclophosphamide and exogenous DNA

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.

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

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.

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

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.

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

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.

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

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.

Molecular genetic characterization of the regulatory region of the Xist gene in the common vole Microtus rossiaemeridionalis

Two conserved regions were identified as a result of interspecific comparison of the 5′-region of the Xist gene, which is the key player in the process of X-chromosome inactivation in mammalian females. The first region corresponds to the minimal promoter, and the second spans between −480 bp and −400 bp from the start of Xist transcription. Footprinting experiments revealed protected regions corresponding to the potential binding sites for TBP, SP1, AP1, SRY, ER, and some other transcription factors. They also demonstrated the interaction with the minimal promoter of the human recombinant transcription factor SP1 in vitro and of the transcription factor CTCF in vivo. Experiments with reporter constructs showed that repressors of Xist transcription were located between −100 bp and −200 bp and between −300 bp and −400 bp and activators of Xist transcription were located between −200 bp and −300 bp and between −400 bp and −500 bp.

A Regulatory Potential of the Xist Gene Promoter in Vole M. rossiaemeridionalis

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.