Evgeniy S. Morozkin

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.

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.

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.

The Effect of Protein Transport Inhibitors on the Production of Extracellular DNA

The presence of various genomic sequences in the pool of extracellular DNA was studied by cloning and sequencing the DNA eluted from the surface of HeLa cells. Sequences of 19 genes, 10 pseudogenes, and 41 repeated elements were found in 103 clones. Sequences of LINE repeats were found in 17% of the clones; consequently real‐time PCR assay for quantification of LINE repeats was chosen to study the influence of protein transport inhibitors on the concentration of free and cell‐surface‐bound DNA in the cultures of HUVEC and HeLa cells. Treatment of both cell lines with the inhibitors did not interfere with the concentration of extracellular DNA in the growth medium, except for chloroquine, which doubled the concentration of extracellular DNA in HUVEC culture. The treatment of HUVECs with monensin, glyburide, or methylamine decreases the cell‐surface‐bound DNA concentration by 30, 35, and 19%, respectively. The incubation of HeLa cells with monensin reduces the concentration of cell‐surface‐bound DNA by 15%; however, the treatment with glyburide increases cell‐surface‐bound DNA concentration by 50%. The data obtained demonstrate the involvement of vesicular transport in generation of extracellular DNA.

A comparative study of cell-free apoptotic and genomic DNA using FISH and massive parallel sequencing

Objective : Study of circulating DNA (cirDNA) generation mechanisms with respect to their influence on the content of cirDNA is very important since it could indicate the best molecular targets for diagnostic applications. Since apoptosis was shown to be one of the main sources of cirDNA, we performed in vitro comparative study of cell-free apoptotic and genomic DNA (gDNA). Methods : DNA isolated from culture medium of apoptotic human umbilical vein endothelial cells (cm-apoDNA) and the gDNA from the same living cells was analyzed using FISH and sequenced on SOLiD 3 platform. Results/conclusions : FISH demonstrates overrepresentation of C-positive chromosome regions in cm-apoDNA. SOLiD 3 data show enrichment of cm-apoDNA for Alu repeats: the content of AluJ, AluS and AluY repeats was, respectively, 2.47-fold (standard deviation (SD) 3.6%), 2.45-fold (SD 5.5%) and 2.79-fold (SD 6.1%) higher in cm-apoDNA. By contrast, some of L1 elements were underrepresented in cm-apoDNA: the content of L1MA and L1ME was, respectively, 1.4-fold (SD 22%) and 1.45-fold (SD 9%) lower in cm-apoDNA. In contrast to FISH, these data and the predominant location of Alu repeats in euchromatic regions evidence the non-uniform gDNA degradation during apoptosis leading to the enrichment of cm-apoDNA with coding sequences.

A phenol-free method for isolation of microRNA from biological fluids.

MicroRNAs (miRNAs) found in biological fluids such as blood and urine have been identified as promising biomarkers for many human disorders, including cancer, cardiopathies, and neurodegenerative diseases. However, circulating miRNAs are either encapsulated into vesicles or found in complexes with proteins and lipoproteins and, thus, require a special approach to their isolation. Acid phenol-chloroform extraction can solve this problem, but it is a labor-intensive procedure that relies heavily on the use of hazardous chemicals. Here we describe a fast and simple phenol-free protocol for miRNA isolation from biofluids. MiRNA is extracted from complexes with biopolymers by a high concentration of guanidine isothiocyanate combined with water/organic composition of solvents. Purification is finished using silica-based spin columns. Comparison of miRNA isolation from blood plasma and urine using the single-phase method and acid phenol-chloroform extraction by means of radioisotope spike-ins and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) showed similar performance of the two methods.

A reliable method to concentrate circulating DNA.

Concentration of circulating DNA probes is required to increase the amount of DNA involved in subsequent study (by polymerase chain reaction, sequencing, and microarray). This work was dedicated to the comparison of five different methods used for concentration of DNA circulating in blood. Precipitation of circulating DNA with acetone in the presence of triethylamine provides minimal DNA loss, high reproducibility, and at least three times higher DNA yield in comparison with the standard ethanol protocol.

Release of Nucleic Acids by Eukaryotic Cells in Tissue Culture

Extracellular nucleic acids in cultures of A431 and HeLa cells were investigated. The data obtained demonstrate the presence of high weight DNA and RNA in the extracellular medium. Temporal changes of extracellular nucleic acids levels in growth medium were investigated.

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.

Comparative Study of Extracellular DNA by FISH

A comparative study of extracellular DNA versus genomic or apoptotic DNA was executed by fluorescent in situ hybridization (FISH) analysis. Extracellular DNA from culture medium and bound to the cell surface of human primary endotheliocytes, human primary fibroblasts and HeLa cells were investigated. There were not any specific peculiarities found in extracellular DNA isolated from the culture medium of endotheliocytes and HeLa cells. We revealed an overrepresentation of chromosome 9 fragments and the regions of the short arms of chromosomes 13, 14, 15, 21, 22 in DNA isolated from the culture medium of primary fibroblasts. The pericentromeric region of chromosome 9 is also overrepresented in cell surface bound DNA isolated from endotheliocytes, fibroblasts and HeLa cells. The data obtained allow a rational selection of DNA targets for the investigation of extracellular DNA generation and circulating DNA-based diagnostics.