Elena Y. Rykova

RARβ2 gene methylation level in the circulating DNA from blood of patients with lung cancer.

Alterations in the patterns of DNA methylation are among the earliest and most common events in tumorigenesis. Epigenetic changes were shown to be detectable in DNA, circulating in blood (cirDNA) of cancer patients, indicating the resources to create the minimally invasive diagnostic tests based on tumor-specific DNA markers. RAR&bgr;2 methylation level was significantly increased in plasma cirDNA and cell surface-bound cirDNA (csb-cirDNA) from patients with non-small cell lung cancer compared with healthy individuals (7620 and 1083 copies/ml in the csb fractions, 3589 and 1068 copies/ml in the blood plasma; P=0.003 and 0.001). The cell-bound-to-cell-free RAR&bgr;2 methylation ratio was found to be elevated in patients with non-small cell lung cancer compared with control (2.12 and 1.01, respectively; P=0.023). RAR&bgr;2 methylation level in csb-cirDNA and plasma cirDNA was higher in stage III patients compared with stage I–II patients (P=0.02 and 0.03). In the subgroup of patients with squamous cell carcinoma, RAR&bgr;2 methylation level in the cbs-cirDNA was higher compared with patients with adenocarcinoma (P=0.04). Epigenetic alterations of tumor suppressor gene RAR&bgr;2 in the total cirDNA (plasma cirDNA and csb-cirDNA) were found to be associated with lung cancer progression. The data obtained indicate that cirDNA-based testing provides a valuable source for subsequent verification of methylated DNA markers for lung cancer diagnostics and prognosis.

Concentrations of circulating RNA from healthy donors and cancer patients estimated by different methods.

Abstract:u2002 Circulating RNA (cirRNA) was isolated from plasma and cell surface‐bound fractions of blood of healthy women and breast cancer patients. RNA samples were DNase treated and quantified by a SYBR Green II assay. Concentrations of RNA sequences of GAPDH, Ki‐67 mRNA, and 18S rRNA were measured by real‐time quantitative PCR (RT‐qPCR) after reverse transcription with random hexamer primers. The obtained data spread over three orders of magnitude for GAPDH and Ki‐67 mRNA signals and two orders of magnitude for the copy number of 18S rRNA in blood fractions in both groups. In blood of healthy donors, no correlation was found between the copy number of GAPDH, Ki‐67 mRNA, and 18S rRNA and RNA concentrations measured by the SYBR Green II assay. Within the group of breast cancer patients, the concentration GAPDH and Ki‐67 mRNA correlated with the concentration of total RNA only in the cell surface‐bound fraction; whereas the concentration of 18S rRNA correlated with total RNA in both, the cell surface‐bound fraction and blood. The copy number of Ki‐67 mRNA correlated with copy numbers of GAPDH mRNA in all fractions of cirRNA of healthy donors and breast cancer patients. A correlation between copy numbers of Ki‐67 mRNA and 18S rRNA was found only in cell surface‐bound fraction of breast cancer patients. The data described here demonstrate the necessity of searching for more suitable RNA markers in order to estimate total cirRNA concentrations by RT‐qPCR, although mRNA of GAPDH could be used for normalization of the level of cancer‐specific mRNA among patients.

Investigation of Tumor‐Derived Extracellular DNA in Blood of Cancer Patients by Methylation‐Specific PCR

The frequency of APC, RASSF1A, RARβ, CDH1 and CDH13 gene promoter methylation in samples of DNA isolated from breast and lung patient plasma was studied in order to develop the noninvasive tumor‐specific DNA detection method. Methylation of at least one of genes was detected in extracellular DNA from most of the cancer blood specimens. The results obtained indicate that promoter hypermethylation of a number of marker genes represents a promising serum marker for early breast and lung cancer detection.

The Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a nuclear docking site for antisense oligonucleotides containing a TAAAT motif

The subcellular localisation of oligodeoxynucleotides (ODN) is a major limitation for their use against nuclear targets. In this study we demonstrate that an antisense ODN directed against cytosolic phospholipase A(2) (cPLA2) mRNA is efficiently taken up and accumulates in the nuclei of endothelial cells (HUVEC), human monocytes and HeLa cells. Gel shift experiments and incubation of cells with oligonucleotide derivatives show that the anti-cPLA2 oligo binds a 37 kDa protein in nuclear extracts. The TAAAT sequence was identified as the major binding motif for the nuclear protein in competition experiments with mutated ODNs. Modification of the AAA triplet resulted in an ODN which failed to localise in the nucleus. Moreover, inserting a TAAAT motif into an ODN localising in the cytosol did not modify its localisation. The 37 kDa protein was purified and identified after peptide sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was shown by confocal microscopy that GAPDH co-localises with anti-cPLA2 ODN in the nucleus and commercial GAPDH effectively binds the oligo. Competition experiments with increasing concentration of NAD(+) co-factor indicate that the GAPDH Rossmann fold is a docking site for antisense oligonucleotides containing a TAAAT motif.

Plasma miR-19b and miR-183 as Potential Biomarkers of Lung Cancer

Lung cancer is a complex disease that often manifests at the point when treatment is not effective. Introduction of blood-based complementary diagnostics using molecular markers may enhance early detection of this disease and help reduce the burden of lung cancer. Here we evaluated the diagnostic potential of seven plasma miRNA biomarkers (miR-21, -19b, -126, -25, -205, -183, -125b) by quantitative reverse transcription PCR. Influence clinical and demographical characteristics, including age, tumor stage and cancer subtype on miRNA levels was investigated. Four miRNAs were significantly dysregulated (miR-19b, -21, -25, -183) in lung cancer patients. Combination of miR-19b and miR-183 provided detection of lung cancer with 94.7% sensitivity and 95.2% specificity (AUC = 0.990). Thus, miRNAs have shown the potential to discriminate histological subtypes of lung cancer and reliably distinguish lung cancer patients from healthy individuals.

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.

Hypomethylation of human-specific family of LINE-1 retrotransposons in circulating DNA of lung cancer patients

Circulating DNA has recently gained attention as a fast and non-invasive way to assess tumor biomarkers. Since hypomethylation of LINE-1 repetitive elements was described as one of the key hallmarks of tumorigenesis, we aimed to establish whether the methylation level of LINE-1 retrotransposons changes in cell-surface-bound fraction of circulating DNA (csbDNA) of lung cancer patients. Methylated CpG Island Recovery Assay (MIRA) coupled to qPCR-based quantitation was performed to assess integral methylation level of LINE-1 promoters in csbDNA of non-small cell lung cancer patients (n=56) and healthy controls (n=44). Deep sequencing of amplicons revealed that hypomethylation of LINE-1 promoters in csbDNA of lung cancer patients is more pronounced for the human-specific L1Hs family. Statistical analysis demonstrates significant difference in LINE-1 promoter methylation index between cancer patients and healthy individuals (ROC-curve analysis: n=100, AUC=0.69, p=0.0012) and supports the feasibility of MIRA as a promising non-invasive approach.

Blood Based Methylated DNA and Tumor-Specific Protein Analysis in Gastric Cancer Diagnostics

Promoter methylation rates of three tumor suppressor genes were detected in the total cirDNA, derived from plasma and cell-surface-bound fractions, of gastric cancer patients (n = 30) and healthy subjects (n = 30) using MS-PCR. The tumor marker protein levels (CEA, CA 72.4, CA 19.9) were assessed in plasma samples by commercial immunoassay kits. Methylated forms of p15, MGMT and hMLH1genes were detected in the total cirDNA with high rates at II, III and IV stages of gastric cancer. Sensitivity of the MS-PCR-based assay for at least one of methylated p15 and hMLH1 genes in gastric cancer was found to be much higher compared with the sensitivity of the immunoassay for elevated levels of CA 72.4 and CA 19.9 proteins (63 and 30%, respectively). No significant correlation was found between epigenetic and protein markers so indicating their independent development in gastric tumor pathogenesis. To conclude, epigenetic alterations of total cirDNA and elevated levels of tumor-associated proteins were found to be independently associated with gastric cancer indicating their usefulness as complementary diagnostic and prognostic markers for gastric cancer.

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE IS RESPONSIBLE FOR INTRANUCLEAR LOCALIZATION OF SOME OLIGONUCLEOTIDES

Nuclear accumulation of ODNs has been associated with their binding to a series of nuclear proteins. These interactions could be responsible for the sequence-independent effects of ODNs as well as for their sequence-specific interactions and their intracellular distribution. Investigation of interaction of ODNs with these proteins may shed light on the mechanisms of cellular uptake and nuclear accumulation of oligonucleotides.