Protease Activity and Cell-Free DNA in Blood Plasma of Healthy Donors and Breast Cancer Patients

Тамкович, С. Н.; Bryzgunova, Olga E.

doi:10.1080/15321819.2015.1069745

Cited by 7 publications

(8 citation statements)

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“…To increase the amount of analyzed tumor DNA, either an increase in venous blood volume is required (which is extremely problematic for patients with advanced stages accompanied by anemia, while there is no increase in the ratio of methylated and unmethylated forms) or the use of more abundant blood-circulating source of extracellular DNA like csbDNA is required. The concentration of cfDNA and their size/fragmentation are determined not only by uneven fragmentation of the genome during cell death, but also by nucleases and proteases that hydrolyze cfDNA hidden in nucleoprotein complexes [ 13 , 14 , 17 ]. These cfDNA features impose various restrictions on both isolation methods and subsequent analysis [ 3 ].…”

Section: Discussionmentioning

confidence: 99%

“…Tumor specific cfDNA was found in short DNA fragments generated by active secretion or the apoptosis of cancer cells, and in longer fragments originating in necrosis and autophagy [ 8 , 9 , 10 , 11 , 12 ]. Due to its origin, cfDNA is fragmented and circulates in the bloodstream as nucleoprotein complexes [ 7 ], and it is additionally degraded by nucleases and proteases in the blood [ 13 , 14 ]. The total hydrolase activity in the blood depends on the concentrations of proenzymes/enzymes and activators/inhibitors [ 15 , 16 ], and may be related to tumor histotype/carcinogenicity.…”

Section: Introductionmentioning

confidence: 99%

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Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy

Тамкович

Tupikin

Kozyakov³

et al. 2022

IJMS

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Aberrantly methylated circulating DNA (cirDNA) has proven to be a good cancer marker, but its detection is limited by low concentrations, fragmentation, and insufficiency. Since the methylated cirDNA was shown to be more stable in circulation than the unmethylated one and was shown to bind with the blood cell surface, we studied the concentration, representation, and fragmentation of tumor-derived methylated DNA in cell-free and cell-surface-associated DNA. We found that long DNA fragments (more than 10 kb) are mainly associated with the surface of blood cells. However, in plasma short DNA fragments (100–1000 bp) were also found along with long DNA fragments. Isolation of short fragments after separation of cirDNA in 6% PAGE followed by quantitative PCR (L1 element) has shown that short DNA fragments in healthy females represent 22% versus 0.5–4.4% in breast cancer patients. The methylated form of the RARβ2 gene was detected only in long DNA fragments by Real-time TaqMan PCR of bisulfite-converted DNA. The methylation index of cirDNA from healthy women was estimated at 0%, 9%, and 7% in plasma, PBS-EDTA, and trypsin eluates from the surface of blood cells, respectively. The methylation index of breast cancer patients’ DNA was found to be 33%, 15%, and 61% in the same fractions confirming the overrepresentation of methylated DNA in csbDNA.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy

Тамкович

Tupikin

Kozyakov³

et al. 2022

IJMS

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“…Cell-free DNA (cfDNA) is detectable in the circulation of healthy people,1–3 and normal dogs 4. In health, this cfDNA originates from various cells, but particularly from death of haematopoietic cells including those of lymphoid and myeloid origins 5 6.…”

Section: Introductionmentioning

confidence: 99%

Effect of sample type on plasma concentrations of cell‐free DNA and nucleosomes in dogs

Goggs

2019

Veterinary Record Open

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Cell-free DNA (cfDNA) and nucleosomes are two biomarkers of cell death and neutrophil extracellular trap formation that are increased in dogs with sepsis, immune-mediated haemolytic anaemia, cancer and following trauma and have diagnostic and prognostic values. cfDNA and nucleosomes are typically measured in plasma samples using DNA-specific fluorophores and ELISA assays, respectively, but their concentrations may be affected by pre-analytical variables such as sample type. The present study aimed to investigate the influence of sample type on the plasma cfDNA and nucleosome concentrations of a heterogeneous group of dogs presenting to an emergency room. Triplicate samples were collected into K2-ethylenediamine tetraacetic acid, 3.2% citrate and a specialised DNA stabilisation tube (Streck BCT), processed rapidly and frozen for batch analysis. Biomarker concentrations were compared between sample types by calculation of Spearman’s correlation coefficients, and with Deming regression, Bland-Altman plots and the Friedman test. Overall, biomarker concentrations were highly correlated between the three sample types. The most concordant results were obtained using citrate samples and the DNA stabilisation tube. Matched cfDNA concentrations between the different sample types were significantly different but there was no significant difference between the nucleosome concentrations in any of the sample types. The present study suggests that cfDNA and nucleosomes can be successfully measured in various sample types, but distinct sample types do not produce interchangeable results. This argues for use of a consistent sample type within studies and suggests standardisation may be useful for the field.

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“…368589) and fractionated into plasma and blood cells within 4 h of blood sampling. Plasma was collected from the blood samples after centrifugation at 400× g for 15 min and 800× g for 20 min and was frozen in aliquots at −20 °C until DNA isolation [ 40 ].…”

Section: Methodsmentioning

confidence: 99%

Locus-Specific Methylation of GSTP1, RNF219, and KIAA1539 Genes with Single Molecule Resolution in Cell-Free DNA from Healthy Donors and Prostate Tumor Patients: Application in Diagnostics

Bryzgunova

Bondar

Ruzankin

et al. 2021

Cancers

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The locus-specific methylation of three genes (GSTP1, RNF219, and KIAA1539 (also known as FAM214B)) in the blood plasma cell-free DNA (cfDNA) of 20 patients with prostate cancer (PCa), 18 healthy donors (HDs), and 17 patients with benign prostatic hyperplasia (BPH) was studied via the MiSeq platform. The methylation status of two CpGs within the same loci were used as the diagnostic feature for discriminating the patient groups. Many variables had good diagnostic characteristics, e.g., each of the variables GSTP1.C3.C9, GSTP1.C9, and GSTP1.C9.T17 demonstrated an 80% sensitivity at a 100% specificity for PCa patients vs. the others comparison. The analysis of RNF219 gene loci methylation allowed discriminating BPH patients with absolute sensitivity and specificity. The data on the methylation of the genes GSTP1 and RNF219 allowed discriminating PCa patients, as well as HDs, with absolute sensitivity and specificity. Thus, the data on the locus-specific methylation of cfDNA (with single-molecule resolution) combined with a diagnostic approach considering the simultaneous methylation of several CpGs in one locus enabled the discrimination of HD, BPH, and PCa patients.

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Protease Activity and Cell-Free DNA in Blood Plasma of Healthy Donors and Breast Cancer Patients

Cited by 7 publications

References 37 publications

Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy

Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy

Effect of sample type on plasma concentrations of cell‐free DNA and nucleosomes in dogs

Locus-Specific Methylation of GSTP1, RNF219, and KIAA1539 Genes with Single Molecule Resolution in Cell-Free DNA from Healthy Donors and Prostate Tumor Patients: Application in Diagnostics

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