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.
Tumour development is characterised by the increased circulating DNA (cirDNA) concentration and by tumour-related changes in blood plasma DNA. Concentration of cirDNA and methylation of RARb2, RASSF1A and HIC-1 gene promoters were investigated in cell-free and cell-surface-bound fractions from healthy donors, patients with breast cancer, and patients with breast fibroadenoma. Tumour development was shown to lead to significant changes in the distribution of cirDNA between cell-free and cell-surfacebound fractions. Analysis of RARb2 and RASSF1A methylation in the total cirDNA provides 95% diagnostic coverage in breast cancer patients, 60% in patients with benign lesions, and is without false-positive results in healthy women. Results of the study indicate that methylation-specific PCR of RARb2 and RASSF1A genes based on the total cirDNA combined with the quantitative analysis of cirDNA distribution between cell-bound and cell-free fractions in blood provide the sensitive and accurate detection and discrimination of malignant and benign breast tumours.
Extracellular nucleic acids are found in different biological fluids in the organism and in the environment: DNA is a ubiquitous component of the organic matter pool in the soil and in all marine and freshwater habitats. Data from recent studies strongly suggest that extracellular DNA and RNA play important biological roles in microbial communities and in higher organisms. DNA is an important component of bacterial biofilms and is involved in horizontal gene transfer. In recent years, the circulating extracellular nucleic acids were shown to be associated with some diseases. Attempts are being made to develop noninvasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. Recent observations demonstrated the possibility of nucleic acids exchange between eukaryotic cells and extracellular space suggesting their participation in so far unidentified biological processes.
Cancer statistics show that recent improvements in cancer management are only mildly effective in the absence of reliable biomarkers for the detection, diagnosis and monitoring of malignant disease. Recently circulating nucleic acids have been suggested as potential biomarker candidates to fill this role. Areas covered: This review focuses on the different types of circulating RNA biomarkers under investigation, describing the latest advances in their development and application to clinical settings, as well as challenges that researchers face in the process. Immediate perspectives of the field are outlined, and authors' recommendations on the best progression path are provided. Expert commentary: The development of RNA-based cancer biomarkers is a thriving area of biomedical research that has progressed significantly over the last decade. However, it seems that it is now at the point, where unless several key issues are resolved, no significant progress can be made further. Currently several areas of biomarker research require re-assessment, as indicated by the latest findings regarding the biology of circulating nucleic acids and the accumulated data of their analysis using various techniques. Additionally, regulating agencies need to be working alongside researchers to facilitate faster and easier adoption of new effective biomarkers into the clinical practice.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.