Pavel P. Laktionov

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.

Methylation‐Specific Sequencing of GSTP1 Gene Promoter in Circulating/Extracellular DNA from Blood and Urine of Healthy Donors and Prostate Cancer Patients

Hypermethylated promoters of cancer‐related genes represent convenient targets for early cancer diagnosis and monitoring based on circulating/extracellular DNA (cir/exDNA) from human blood and urine. The frequency of detection of methylated tumor suppressor genes in plasma or urine samples is usually lower than in the samples of tumor tissue because of a low concentration of target DNA and potential polymorphism of cirDNA methylation. Sequencing of the methylated cir/exDNA of tumor suppression genes provides information about methylation of the cirDNA originating from the tumor cells, which is necessary for optimization of cancer diagnosis. In this work, by sequencing chemically converted cir/exDNA, we have studied the cytosine methylation profile of GSTP1 gene promoter (1001–1302, X08058) in the pool of cir/exDNA from the blood and urine of healthy men, prostate cancer (PCa) patients, and patients with benign prostatic hyperplasia (BPH). We demonstrated that the data on cir/exDNA methylation could be obtained from sequencing of the cir/exDNA from blood and urine. The DNA isolated from blood plasma and the eluates of blood cells and urine of each patient were characterized by the same methylation profile of the GSTP1 gene. The profile of GSTP1 gene methylation in the extracellular DNA of PCa patients differs from the profiles characteristic of healthy donors and patients with BPH.

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.

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.

Epigenetic markers of prostate cancer in plasma circulating DNA

Epigenetic differences are a common feature of many diseases, including cancer, and disease-associated changes have even been detected in bodily fluids. DNA modification studies in circulating DNA (cirDNA) may lead to the development of specific non-invasive biomarkers. To test this hypothesis, we investigated cirDNA modifications in prostate cancer patients with locally confined disease (n = 19), in patients with benign prostate hyperplasias (n = 20) and in men without any known prostate disease (n = 20). This initial discovery screen identified 39 disease-associated changes in cirDNA modification, and seven of these were validated using the sodium bisulfite-based mapping of modified cytosines in both the discovery cohort and an independent 38-patient validation cohort. In particular, we showed that the DNA modification of regions adjacent to the gene encoding ring finger protein 219 distinguished prostate cancer from benign hyperplasias with good sensitivity (61%) and specificity (71%). We also showed that repetitive sequences detected in this study were meaningful, as they indicated a highly statistically significant loss of DNA at the pericentromeric region of chromosome 10 in prostate cancer patients (p = 1.8 × 10(-6)). Based on these strong univariate results, we applied machine-learning techniques to develop a multi-locus biomarker that correctly distinguished prostate cancer samples from unaffected controls with 72% accuracy. Lastly, we used systems biology techniques to integrate our data with publicly available DNA modification and transcriptomic data from primary prostate tumors, thereby prioritizing genes for further studies. These data suggest that cirDNA epigenomics are promising source for non-invasive biomarkers.

Comparative Study of Extracellular Vesicles from the Urine of Healthy Individuals and Prostate Cancer Patients.

Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90–95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.