Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell
Objective. Easily oxidizable GC-rich DNA (GC-DNA) fragments accumulate in the cell-free DNA (cfDNA) of patients with various diseases. The human oxidized DNA penetrates the MCF7 breast cancer cells and significantly changes their physiology. It can be assumed that readily oxidizable GC-DNA fragments...
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2019-01-01
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Series: | Oxidative Medicine and Cellular Longevity |
Online Access: | http://dx.doi.org/10.1155/2019/2348165 |
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doaj-5ded6fd9a1d14ea4aaf53520d84858ab2020-11-25T00:47:37ZengHindawi LimitedOxidative Medicine and Cellular Longevity1942-09001942-09942019-01-01201910.1155/2019/23481652348165Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer CellSvetlana V. Kostyuk0Nadezhda N. Mordkovich1Natalya A. Okorokova2Vladimir P. Veiko3Elena M. Malinovskaya4Elizaveta S. Ershova5Marina S. Konkova6Ekaterina A. Savinova7Maria A. Borzikova8Tatiana A. Muzaffarova9Lev N. Porokhovnik10Nataly N. Veiko11Serguey I. Kutsev12Research Centre for Medical Genetics (RCMG), Moscow 115478, RussiaBach Institute of Biochemistry, Biotechnology Research Center, Russian Academy of Sciences, Moscow 119071, RussiaBach Institute of Biochemistry, Biotechnology Research Center, Russian Academy of Sciences, Moscow 119071, RussiaBach Institute of Biochemistry, Biotechnology Research Center, Russian Academy of Sciences, Moscow 119071, RussiaResearch Centre for Medical Genetics (RCMG), Moscow 115478, RussiaResearch Centre for Medical Genetics (RCMG), Moscow 115478, RussiaResearch Centre for Medical Genetics (RCMG), Moscow 115478, RussiaResearch Centre for Medical Genetics (RCMG), Moscow 115478, RussiaResearch Centre for Medical Genetics (RCMG), Moscow 115478, RussiaResearch Centre for Medical Genetics (RCMG), Moscow 115478, RussiaResearch Centre for Medical Genetics (RCMG), Moscow 115478, RussiaResearch Centre for Medical Genetics (RCMG), Moscow 115478, RussiaResearch Centre for Medical Genetics (RCMG), Moscow 115478, RussiaObjective. Easily oxidizable GC-rich DNA (GC-DNA) fragments accumulate in the cell-free DNA (cfDNA) of patients with various diseases. The human oxidized DNA penetrates the MCF7 breast cancer cells and significantly changes their physiology. It can be assumed that readily oxidizable GC-DNA fragments can penetrate the cancer cells and be expressed. Methods. MCF7 cells were cultured in the presence of two types of GC-DNA probes: (1) vectors pBR322 and pEGFP and (2) plasmids carrying inserted human rDNA (pBR322-rDNA and pEGFP-rDNA). pEGFP and pEGFP-rDNA contained a CMV promoter and a fluorescent protein gene EGFP. ROS generation rate, accumulation of the DNA probes in MCF7, 8-oxodG content, expression of EGFP and NOX4, and localization of EGFP, NOX4, and 8-oxodG in MCF7 were explored. The applied methods were qPCR, fluorescent microscopy (FM), immunoassay, and flow cytometry (FCA). Results. When GC-DNA is added to the cell culture medium, it interacts with the cell surface. At the site of GC-DNA contact with the cell, NOX4 is expressed, and ROS level increases. The ROS oxidize the GC-DNA. When using the plasmids pEGFP and pEGFP-rDNA, an increase in the amount of the DNA EGFP, RNA EGFP, and EGFP proteins was detected in the cells. These facts suggest that GC-DNA penetrates the cells and the EGFP gene is expressed. Insertions of the rDNA significantly increase the GC-DNA oxidation degree as well as the rate of plasmid transfection into the cells and the EGFP expression level. In the nucleus, the oxidized GC-rDNA fragments, but not the vectors, are localized within the nucleolus. Conclusions. GC-rich cfDNA fragments that are prone to oxidation can easily penetrate the cancer cells and be expressed. The cfDNA should become a target for the antitumor therapy.http://dx.doi.org/10.1155/2019/2348165 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Svetlana V. Kostyuk Nadezhda N. Mordkovich Natalya A. Okorokova Vladimir P. Veiko Elena M. Malinovskaya Elizaveta S. Ershova Marina S. Konkova Ekaterina A. Savinova Maria A. Borzikova Tatiana A. Muzaffarova Lev N. Porokhovnik Nataly N. Veiko Serguey I. Kutsev |
spellingShingle |
Svetlana V. Kostyuk Nadezhda N. Mordkovich Natalya A. Okorokova Vladimir P. Veiko Elena M. Malinovskaya Elizaveta S. Ershova Marina S. Konkova Ekaterina A. Savinova Maria A. Borzikova Tatiana A. Muzaffarova Lev N. Porokhovnik Nataly N. Veiko Serguey I. Kutsev Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell Oxidative Medicine and Cellular Longevity |
author_facet |
Svetlana V. Kostyuk Nadezhda N. Mordkovich Natalya A. Okorokova Vladimir P. Veiko Elena M. Malinovskaya Elizaveta S. Ershova Marina S. Konkova Ekaterina A. Savinova Maria A. Borzikova Tatiana A. Muzaffarova Lev N. Porokhovnik Nataly N. Veiko Serguey I. Kutsev |
author_sort |
Svetlana V. Kostyuk |
title |
Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell |
title_short |
Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell |
title_full |
Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell |
title_fullStr |
Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell |
title_full_unstemmed |
Increased Transfection of the Easily Oxidizable GC-Rich DNA Fragments into the MCF7 Breast Cancer Cell |
title_sort |
increased transfection of the easily oxidizable gc-rich dna fragments into the mcf7 breast cancer cell |
publisher |
Hindawi Limited |
series |
Oxidative Medicine and Cellular Longevity |
issn |
1942-0900 1942-0994 |
publishDate |
2019-01-01 |
description |
Objective. Easily oxidizable GC-rich DNA (GC-DNA) fragments accumulate in the cell-free DNA (cfDNA) of patients with various diseases. The human oxidized DNA penetrates the MCF7 breast cancer cells and significantly changes their physiology. It can be assumed that readily oxidizable GC-DNA fragments can penetrate the cancer cells and be expressed. Methods. MCF7 cells were cultured in the presence of two types of GC-DNA probes: (1) vectors pBR322 and pEGFP and (2) plasmids carrying inserted human rDNA (pBR322-rDNA and pEGFP-rDNA). pEGFP and pEGFP-rDNA contained a CMV promoter and a fluorescent protein gene EGFP. ROS generation rate, accumulation of the DNA probes in MCF7, 8-oxodG content, expression of EGFP and NOX4, and localization of EGFP, NOX4, and 8-oxodG in MCF7 were explored. The applied methods were qPCR, fluorescent microscopy (FM), immunoassay, and flow cytometry (FCA). Results. When GC-DNA is added to the cell culture medium, it interacts with the cell surface. At the site of GC-DNA contact with the cell, NOX4 is expressed, and ROS level increases. The ROS oxidize the GC-DNA. When using the plasmids pEGFP and pEGFP-rDNA, an increase in the amount of the DNA EGFP, RNA EGFP, and EGFP proteins was detected in the cells. These facts suggest that GC-DNA penetrates the cells and the EGFP gene is expressed. Insertions of the rDNA significantly increase the GC-DNA oxidation degree as well as the rate of plasmid transfection into the cells and the EGFP expression level. In the nucleus, the oxidized GC-rDNA fragments, but not the vectors, are localized within the nucleolus. Conclusions. GC-rich cfDNA fragments that are prone to oxidation can easily penetrate the cancer cells and be expressed. The cfDNA should become a target for the antitumor therapy. |
url |
http://dx.doi.org/10.1155/2019/2348165 |
work_keys_str_mv |
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