Elena Yu. Rykova

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.

Cell-surface-bound nucleic acids: Free and cell-surface-bound nucleic acids in blood of healthy donors and breast cancer patients.

Abstract: Concentrations of extracellular DNA and RNA in the blood of healthy donors and patients with malignant and nonmalignant breast tumors were investigated. Cell‐surface‐bound extracellular DNA and RNA were detached by PBS‐EDTA treatment or mild trypsin treatment of erythrocytes and leukocytes. In healthy donors, almost all extracellular nucleic acids (98%) are bound at the surface of blood cells. In the blood of cancer patients, extracellular nucleic acids were found in plasma and not at the cell surface. In patients with nonmalignant breast tumors, extracellular nucleic acids were found both at the surface of blood cells and in plasma. In healthy donors, the cell‐surface‐bound DNA is represented by 20‐kbp DNA fragments and smaller fragments that varied in amounts in different fractions.

Isolation and Comparative Study of Cell-Free Nucleic Acids from Human Urine

Abstract:  Cell‐free nucleic acids (NA) from human urine were investigated. Concentrations of DNA and RNA in the urine of healthy people were independent of gender and were in the range of 6 ng/mL to 50 ng/mL and 24 ng/mL to 140 ng/mL, respectively. DNA fragments of 150–400 bp represent the main part of cell‐free DNA, along with DNA fragments up to 1,300 bp, which were found in male urine, and DNA fragments up to 19 kbp, which were found in female urine. Analysis of circulating DNA, isolated from blood of breast cancer patients and cell‐free DNA isolated from their urine by methylation‐specific PCR, demonstrates that the presence of methylated promoters of RASSF1A and RARβ2 genes in plasma was accompanied by the detection of the same methylated markers in urine. The data obtained demonstrate applicability of cell‐free urine DNA in cancer diagnostics.

Cell-free and cell-bound circulating nucleic acid complexes: mechanisms of generation, concentration and content

Introduction: Extracellular nucleic acids are found in human blood and cell culture medium as cell-free or being adsorbed at cell surface. In the last years, the circulating extracellular nucleic acids in blood were shown to be associated with certain diseases. Attempts are made to develop non-invasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. Areas covered: This article reviews accumulating data regarding cell-free and cell-surface-bound extracellular nucleic acid nature and generation mechanisms. Their existence as a constituent of the naturally occurring complexes with proteins or membrane-bearing particles is discussed with regard to their homeostatic concentration and distribution in healthy donor blood which are significantly altered in cancer patients. Gene-target and whole-genome studies reveal significant differences in gene representation between extracellular DNA and genome DNA. Overrepresentation of regions with high transcription activity has led to proposal that extracellular DNA generation is strongly dependent on the parent genome functionality, which is associated with chromosome packaging and DNA methylation levels. Expert opinion: Recent studies provide evidence of the circulating nucleome organization complexity indicating that discovery of extracellular DNA generation and circulation patterns in healthy condition and cancer is essential to enable the development of proper approaches for the selection of valid diagnostic markers.

Circulating DNA in the Blood of Gastric Cancer Patients

The concentration of cell‐free DNA and promoter methylation status of the MGMT, p15, and hMLH1 genes were analyzed by a fluorescence‐based assay and methylation‐specific PCR (MSP) in the blood of gastric cancer patients (n= 20) and healthy subjects (n= 22). Gastric cancer patients were characterized by an increased concentration of circulating DNA in the plasma; the amount of cell‐surface‐bound DNA was not decreased compared with controls and amounted to 80 ± 15% of the total circulating DNA. MSP analysis of three genes in the cell‐surface‐bound DNA permits the detection of gastric cancer patients with a sensitivity of 75% and a specificity of 54%. Thus, the cell‐surface‐bound DNA is a convenient source of DNA for MSP analysis of cancer‐specific markers. The data on the presence of methylated DNA in plasma combined with the analysis of other cancer‐related changes in DNA can significantly contribute to cancer diagnostics.

Extracellular Circulating Nucleic Acids in Human Plasma in Health and Disease

The concentration of extracellular DNA and RNA in blood plasma of healthy donors, trauma patients, patients with breast and lung cancer, nonmalignant breast tumors and nonmalignant lung diseases were estimated. Significant amounts of extracellular RNA were found in plasma of trauma patients. The concentration of DNA and RNA in plasma of trauma patients correlates with the extent of posttraumatic organ failure. Extracellular RNA was not found in the plasma of breast cancer patients and patients with nonmalignant breast tumors, whereas a very high concentration of extracellular RNA was found in patients with malignant and nonmalignant diseases of lung.

Extracellular nucleic acids in cultures of long-term cultivated eukaryotic cells

Abstract: Investigation of the kinetics of nucleic acid release by HeLa (human cervical carcinoma cell line) and A431 (human squamous carcinoma cell line) cells is presented. The released DNA and RNA were shown to accumulate in culture medium and at the cell surface. A portion of cell surface bound RNA can be eluted with PBS/EDTA. Mild trypsin treatment is required for complete detachment of cell surface bound RNA and cell surface bound DNA. Electrophoretic analysis reveals characteristic patterns of cell‐associated and free RNA and DNA molecules.

Extracellular DNA in breast cancer: Cell-surface-bound, tumor-derived extracellular DNA in blood of patients with breast cancer and nonmalignant tumors.

Abstract: A methylation‐specific polymerase chain reaction technique was used to investigate aberrant promoter methylation of RASSF1A and HIC‐1 genes in circulating extracellular DNA (exDNA) from the blood of breast cancer and fibroadenoma patients. Methylated DNA could be detected in the exDNA eluted from the surface of erythrocytes and leukocytes, even in the samples where no methylated DNA could be detected in plasma. The data obtained demonstrate that cell surface bound exDNA provides a valuable source of material for early noninvasive cancer diagnostics and monitoring.

Cell-Surface-Bound Circulating DNA as a Prognostic Factor in Lung Cancer

Since the mortality of lung cancer patients remains very high, development of prognostic methods essential for efficient therapy is an immediate task. This study was designed to assess the value of circulating DNA (cirDNA) in blood as a prognostic marker in patients with non–small cell lung cancer. The average concentration of cirDNA in plasma was shown to be similar in healthy donors and lung cancer patients. However, the concentration of cell‐surface‐bound circulating DNA (csb‐cirDNA) in lung cancer patients is significantly lower than that found in healthy donors (P < 0.0001) and correlates with a poor prognosis of tumor disease. Quantification of the cell‐surface‐bound DNA in blood of untreated patients allows persons with a poor prognosis of tumor disease to be detected with 94% sensitivity and 50% specificity.

Simple and rapid procedure suitable for quantitative isolation of low and high molecular weight extracellular nucleic acids.

The procedure based on binding of nucleic acids with glass surface in presence of chaotropic salts was adapted for efficient isolation of 100–10000 b.p. DNA fragments and 50–10,000 b. RNA fragments. The method provide 90% and 85% efficacy of isolation of 100 b.p. DNA and 100 b. RNA fragments respectively. High molecular weight nucleic acids are isolated with 98% efficacy. Isolated nucleic acids are free from contaminations, influencing nucleic acids modifying enzymes and fluorochromes. The method is rapid, simple and cost‐effective.