Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell

Kostyuk, Svetlana V.; Mordkovich, N. N.; Okorokova, N. A.; Вейко, В. П.; Malinovskaya, Elena M.; Ershova, Elizaveta S.; Konkova, Marina S.; Savinova, Ekaterina A.; Borzikova, Maria; Музаффарова, Т. А.; Porokhovnik, Lev N.; Вейко, Н. Н.; Kutsev, Serguey I.

doi:10.1155/2019/2348165

Cited by 9 publications

(14 citation statements)

References 35 publications

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“…apoptosis levels, glucose consumption rate, proliferation rate, cell cycle phase, etc.). 4,[11][12][13][14][15] Consequently, the concentration and size of cfDNA measured at any instance may differ significantly for a single cell line and between cell lines. For example, some cell lines may be characterized by a low concentration of high molecular weight cfDNA, while others may be characterized by a high concentration of low molecular weight DNA.…”

Section: Resultsmentioning

confidence: 99%

Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant

2019

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Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA-based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cellfree DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop One c . Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.

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Section: Resultsmentioning

confidence: 99%

Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant

2019

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“…Previously, we have shown that the ability of oxidatively damaged cfDNA to be transferred into human cancer and stem cells significantly depends on their oxidative modifications [14, 24, 25, 27] and identified signaling pathways, activated by oxidized cfDNA in different human cells [14, 18, 25, 31]. We suggested that oxidized cfDNA, produced by dying cells under endogenous or exogenous oxidative stress, is able to be transferred into neurons and glial cells, activating signaling pathways genes.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, we have shown that oxidized cfDNA penetration into the cells is accompanied by the induction of the reactive oxygen species (ROS) synthesis [14, 24, 25, 27] that may lead to the oxidatively generated DNA modifications in cultured cells. An addition of oxidized cfDNA fragments (concentration 50 ng/mL, cultivation time 2 h) to the culture medium increases the 8-oxodG level in cultured cells by 5-6 times ( p < 0.001; Figure 3(a)).…”

Section: Resultsmentioning

confidence: 99%

“…Such biological role of cfDNA signaling is associated with signaling about damage or any other adverse effects that induce oxidative stress and consequent cell death [23]. Previously, we showed that during oxidative stress oxidized cfDNA may be transferred into the cancer and stem cells [24, 25]. Transfer of oxidized cfDNA into the cells leads to the induction of the free radical production, oxidation of nuclear and mitochondrial DNA, and antioxidant protection of cells through activation of а master regulator of antioxidant system—NRF2 enhancement [14, 24–27].…”

Section: Introductionmentioning

confidence: 99%

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Oxidized Cell-Free DNA Role in the Antioxidant Defense Mechanisms under Stress

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Shmarina

Ershova

et al. 2019

Oxidative Medicine and Cellular Longevity

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The present study focuses on the investigation of the oxidized cell-free DNA (cfDNA) properties in several experimental models, including cultured cerebellum cells, peripheral blood lymphocytes (PBL), plasma, and hippocampus under an acute and chronic unpredictable stress model in rats. Firstly, our study shows that Spectrum Green fluorescence-labeled oxidized cfDNA fragments were transferred into the cytoplasm of 80% of the cerebellum culture cells; meanwhile, the nonoxidized cfDNA fragments do not pass into the cells. Oxidized cfDNA stimulates the antioxidant mechanisms and induction of transcription factor NRF2 expression, followed by an activation of NRF2 signaling pathway genes—rise of Nrf2 and Hmox1 gene expression and consequently NRF2 protein synthesis. Secondly, we showed that stress increases plasma cfDNA concentration in rats corresponding with the duration of the stress exposure. At the same time, our study did not reveal any significant changes of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) level in PBL of rats under acute or chronic stress, probably due to the significantly increased Nrf2 expression, that we found in such conditions. 8-oxodG is one of the most reliable markers of DNA oxidation. We also found an increased level of 8-oxodG in the hippocampal homogenates and hippocampal dentate gyrus in rats subjected to acute and chronic stress. Taken together, our data shows that oxidized cfDNA may play a significant role in systemic and neuronal physiological mechanisms of stress and adaptation.

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“…A question whether plasmid DNA requires linearization before adding to the culture medium was considered earlier (45). Our tests failed to show any difference between the rates of accumulation of intact GC-DNAs and their linearized forms in the cells.…”

Section: Methodsmentioning

confidence: 99%

Ribosomal DNA as DAMPs Signal for MCF7 Cancer Cells

et al. 2019

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Introduction: The cell free ribosomal DNA (cf-rDNA) is accrued in the total pool of cell free DNA (cfDNA) in some non-cancer diseases and demonstrates DAMPs characteristics. The major research questions: (1) How does cell free rDNA content change in breast cancer; (2) What type of response in the MCF7 breast cancer cells is caused by cf-rDNA; and (3) What type of DNA sensors (TLR9 or AIM2) is stimulated in MCF7 in response to the action of cf-rDNA? Materials and Methods: CfDNA and gDNA were isolated from the blood plasma and the cells derived from 38 breast cancer patients and 20 healthy female controls. The rDNA content in DNA was determined using non-radioactive quantitative hybridization. In order to explore the rDNA influence on MCF7 breast cancer cells, the model constructs (GC-DNAs) were applied: pBR322-rDNA plasmid (rDNA inset 5836 bp long) and pBR322 vector. ROS generation, DNA damage, cell cycle, expression of TLR9, AIM2, NF-kB, STAT3, and RNA for 44 genes affecting the cancer cell viability were evaluated. The methods used: RT-qPCR, fluorescent microscopy, immunoassay, flow cytometry, and siRNA technology. Results: The ratio R = cf-rDNA/g-rDNA for the cases was higher than for the controls (median 3.4 vs. 0.8, p < 10 −8 ). In MCF7, GC-DNAs induce a ROS burst, DNA damage response, and augmentation of NF-kB and STAT3 activity. The number of the apoptotic cells decreases, while the number of cells with an instable genome (G2/M– arrest, micronuclei) increase. Expression of anti-apoptotic genes ( BCL2, BCL2A1, BCL2L1, BIRC3, MDM2 ) is elevated, while expression of pro-apoptotic genes ( BAX, BID, BAD, PMAIP1, BBC3 ) is lowered. The cells response for pBR322-rDNA is much more intense and develops much faster, than response for pBR322, and is realized through activation of TLR9- MyD88 - NF-kB- signaling. This difference in response speed is owing to the heightened oxidability of pBR322-rDNA and better ability to penetrate the cell. Induction of TLR9 expression in MCF7 is followed by blocking AIM2 expression. Conclusion: (1) Ribosomal DNA accumulates in cfDNA of breast cancer patients; (2) Cell free rDNA induce DNA damage response and stimulates cells survival, including cells with an instable genome; (3) Cell free rDNA triggers TLR9- MyD88- NF-kB- signaling, with significantly repressing the expression of AIM2.

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Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell

Cited by 9 publications

References 35 publications

Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant

Comparison of methods for the quantification of cell-free DNA isolated from cell culture supernatant

Oxidized Cell-Free DNA Role in the Antioxidant Defense Mechanisms under Stress

Ribosomal DNA as DAMPs Signal for MCF7 Cancer Cells

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