Anna V. Cherepanova

Circulating DNA and DNase Activity in Human Blood

Abstract:  The concentration of circulating DNA (cirDNA) and deoxyribonuclease activity in blood plasma of healthy donors and patients with colon or stomach cancer were analyzed. The concentration of DNA was measured using Hoechst 33258 fluorescent assay after the isolation by the glass–milk protocol. A 1‐kbp PCR product labeled with biotinylated forward and fluorescein‐labeled reverse primers was used as a substrate for DNase. DNase activity was estimated from the data of immunochemical detection of the nonhydrolyzed amplicon. The average concentration of cirDNA in the plasma of healthy donors was low (34 ± 34 ng/mL), and was accompanied with high DNase activity (0.356 ± 0.410 U/mL). The increased concentrations of cirDNA in blood plasma of patients with colon and stomach cancer were accompanied by a decrease in DNase activity below the detection level of the assay. The data obtained demonstrate that low DNase activity in blood plasma of cancer patients can cause an increase in the concentration of cirDNA.

Deoxyribonuclease Activity and Circulating DNA Concentration in Blood Plasma of Patients with Prostate Tumors

The DNase activity and circulating DNA (cirDNA) concentration in blood plasma of healthy donors, patients with chronic prostatitis, and patients with prostate tumors were analyzed. The concentration of the cirDNA from plasma was determined by PicoGreen fluorescent assay. DNase activity in blood was measured using the immunoassay based on the cleavage of a hapten‐labeled 974‐bp DNA substrate. The mean cirDNA concentration in the plasma of healthy donors was low (21 ± 4 ng/mL total blood) and was accompanied by high DNase activity (0.17 ± 0.04 U/mL blood). The mean cirDNA concentration was 90 ng/mL blood (10–234 ng/mL) in the patients with nonmalignant prostate tumors and 115 ng/mL blood (13–339 ng/mL) in those with prostate cancer. The mean DNase activity in blood plasma of the patients with prostate tumors was 0.06 U/mL blood (0–0.12 U/mL). The results obtained demonstrate that increased concentrations of cirDNA in blood of the patients with prostate tumors is accompanied by a decreased DNase activity, confirming our previous data that a low DNase activity in blood plasma of cancer patients is one reason for a high cirDNA concentration.

Triterpenoid saponins from the roots of Acanthophyllum gypsophiloides Regel

Summary Two new triterpenoid saponins 1 and 2 were isolated from the methanol extract of the roots of Acanthophyllum gypsophiloides Regel. These saponins have quillaic acid or gypsogenin moieties as an aglycon, and both bear similar sets of two oligosaccharide chains, which are 3-O-linked to the triterpenoid part trisaccharide α-L-Arap-(1→3)-[α-D-Galp-(1→2)]-β-D-GlcpA and pentasaccharide β-D-Xylp-(1→3)-β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[β-D-Quip-(1→4)]-β-D-Fucp connected through an ester linkage to C-28. The structures of the obtained saponins were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy. A study of acute toxicity, hemolytic, anti-inflammatory, immunoadjuvant and antifungal activity was carried out. Both saponins 1 and 2 were shown to exhibit immunoadjuvant properties within the vaccine composition with keyhole limpet hemocyanin-based immunogen. The availability of saponins 1 and 2 as individual pure compounds from the extract of the roots of A. gypsophiloides makes it a prospective source of immunoactive agents.

Concentration and Distribution of Single-Copy β -Actin Gene and LINE-1 Repetitive Elements in Blood of Lung Cancer Patients

The concentration of circulating DNA (cirDNA) in blood plasma and cell-surface-bound fractions of lung cancer patients and healthy individuals was measured using real-time PCR for the single-copy β-actin gene and LINE-1 repetitive elements. The average concentration of cirDNA in plasma was shown to be similar in healthy individuals and non-small cell lung cancer (NSCLC) patients. However, the concentration of cell-surface-bound circulating DNA (csb-cirDNA) in NSCLC patients was significantly lower than that found in healthy individuals (P = 0.009 and P = 0.002 for β-actin and LINE-1 assays, respectively). The decrease of csb-cirDNA concentration in NSCLC patients was associated with a poor disease prognosis. The ratio of the β-actin gene to LINE-1 fragments in the csb-cirDNA was found to be elevated in NSCLC patients compared with control (3.4 and 1.7 respectively, P = 0.007). Thus, in lung cancer patients the cirDNA quantification by PCR for β-actin gene and LINE-1 fragments was found to provide a subsidiary data for tumor detection and prognosis.

Blood Deoxyribonuclease Activity in Health and Diseases

The review summarizes literature data on (deoxy)ribonuclease activity in blood of healthy donors and patients with diseases. Special attention is paid to methodological aspects of the analysis of deoxyribonuclease activity and factors, which interfere with enzyme activity in blood.

Deoxyribonuclease activity in biological fluids of healthy donors and cancer patients.

Integral activity of neutral deoxyribonucleases in the plasma and urine or donors, patients with benign prostatic hyperplasia, and patients with stomach and prostatic cancer was studied by IFA based on hydrolysis of DNA fragment modified with haptene molecules. In donors plasma deoxyribonuclease activity was 0.16±0.04, urinary activity 1.49±1.41 act. U/ml. In patients with benign prostatic hyperplasia and malignant tumors the integral activity of blood deoxyribonucleases was significantly below the normal, and in tumors it did not correlate with tumor size and disease stage. A significant correlation between blood and urinary deoxyribonuclease activities was detected.

Cell-Surface-Bound DNA Inhibits Poly(I:C)-Activated IL-6 and IL-8 Production in Human Primary Endothelial Cells and Fibroblasts

The effects of cell-surface-bound extracellular DNA (csbDNA) on interleukin production by primary human umbilical vein endothelial cells and gingival fibroblasts were investigated. DNA concentrations were measured by Pico Green assay after isolation. Extracellular DNA as well as genomic DNA does not influence IL-6 and IL-8 production in a broad range of concentrations. Simultaneous stimulation of cells with both DNA and TLR3 ligand poly(I:C) leads to inhibition of poly(I:C) activated secretion of IL-6 and IL-8. An almost three times stronger inhibiting effect by cell-surface-bound exDNA as compared to genomic DNA demonstrates the enrichment of cell-surface-bound DNA with immuno-inhibiting sequences and their potential role as antiviral immune response regulators.

DNA inhibits dsRNA-induced secretion of pro-inflammatory cytokines by gingival fibroblasts.

Nucleic acids interacting with pattern-recognizing receptors (PRRs), such as Toll-like-(TLRs), RIG-I-like receptors (RLRs) and dsDNA-receptors activate innate immune response in non-professional immune cells and thus the production of pro-inflammatory cytokines. Along with bacterial and viral nucleic acids, endogenous cell-free and cell-surface-bound extracellular DNA (exDNA and csbDNA) could interact with PRRs and possess immunomodulating activity. To elucidate if exDNA influence innate immunity a comparative study of exDNA, genomic and plasmid DNA on interleukin production in gingival fibroblasts (GF) has been done. All DNA tested have no effect on IL secretion in a broad concentration range (10 ng/ml-1 μg/ml). Simultaneous treatment of cells with DNA and dsRNA analog poly(I:C) leads to inhibition of poly(I:C)-activated secretion of IL-6 and IL-8. Cell-surface-bound DNA possesses two times stronger inhibiting effect as compared to genomic DNA indicating the enrichment of csbDNA in sequences providing such activity. Effects of several recently found specific DNA sequences tightly bound with cell surface have been tested. Joint stimulation of GF with poly(I:C) and deoxyribooligonucleotides (ODN), containing such sequences, demonstrates that both ssODN and dsODN possess sequence-dependent inhibiting effect. Inhibition of IL production after colipofection of ODN and poly(I:C) into cells indicates the involvement of RLRs or other cytoplasmic factors in the effect. The data obtained indicate that endogenous DNA might be involved in regulation of antiviral immune response and sequence-specific ODNs are potential inhibitors of the inflammation induced by viral infection.

Ku protein as the main cellular target of cell-surface-bound circulating DNA

Objective: An immunomodulatory activity of circulating DNA (cirDNA) is implemented via the interactions of cirDNA with the targets exposed on the cell membrane and/or intracellular targets. The goal of this work was to identify the cellular targets of immunoinhibiting cell-surface-bound cirDNA (csbDNA) using its oligodeoxyribonucleotide (ODN) analogs containing the nucleotide motifs frequently found in csbDNA and displaying the same effects. Materials and methods: The binding of [32P]-labeled single- and double-stranded ODNs (ss- and ds-ODNs) with membrane–cytosolic (MC) extracts and living human umbilical vein endothelial cells (HUVEC) was studied by electromobility shift assay (EMSA). Complexes of biotinylated ODNs with target proteins were affinity isolated using streptavidin Sepharose with subsequent SDS-PAGE and identified by MALDI-TOF mass spectrometry. Results and conclusions: Both ss- and ds-ODNs form strong ODN–protein complexes with similar electrophoretic mobilities after incubation with the MC extracts of HUVEC either when added extracellularly or lipofected into cells. The ODN-binding proteins were identified as the DNA-binding components of DNA-dependent protein kinase (DNA-PK), namely, Ku70 and Ku80 proteins. Diverse cellular localizations and functions of the Ku proteins demand further clarification of Ku70/80 role as a mediator of the csbDNA immunoinhibiting effects.

Oligodeoxynucleotide Analogues of Circulating DNA Inhibit dsRNA-Induced Immune Response at the Early Stages of Signal Transduction Cascade in a Cell Type-Dependent Manner

Oligodeoxynucleotide (ODN) analogues of cell-surface-bound circulating DNA inhibit the dsRNA-induced production of pro-inflammatory interleukin 6, interferon beta and antibacterial peptide beta-defensin 2 not only in human gingival fibroblasts, but also in human primary endothelial and transformed cells (Hela and A431). ODN analogues do not effect dendritic cells activation by poly(I:C). The data obtained indicate that the early stages of the signal transduction cascade are violated by ODN analogues and the effects depend on the cell type.