Extracellular DNA Containing (dG)n Motifs Penetrates into MCF7 Breast Cancer Cells, Induces the Adaptive Response, and Can Be Expressed

Abstract

Background Oxidized human DNA or plasmid DNAs containing human ribosomal genes can easily penetrate into the breast cancer cells MCF7 and stimulate the adaptive response induction. Plasmid DNA containing a CMV promoter, gene EGFP, and the insertion of the human ribosomal genes can be expressed. A hypothesis is proposed: these features of the ribosomal DNA are due to the presence of dGn motifs that are prone to oxidize. Methods Cells of MCF7 line were cultured with plasmids which contained a CMV promoter and gene of fluorescent protein EGFP. Genetic construction pEGFP-Gn contains pEGFP vector and a small insertion with dG11 and dG13 motifs that are inclined to oxidation. The accumulation of pEGFP and pEGFP-Gn in MCF7 (qPCR), the levels of ROS in the cells, the content of 8-oxodG in plasmids and cellular DNA (flow cytometry, immunoassay, and fluorescent microscopy), the expression of NOX4 and EGFP, the localization of NOX4 and EGFP in MCF7 (qPCR, flow cytometry, and fluorescent microscopy), and the levels of the cell DNA damage (comet assay) were analyzed. Results (dG)n insertions in the plasmid pEGFP increase the levels of ROS, the cell DNA oxidation and DNA damage, and the level of transfection of plasmid into the MCF7 cells. NOX4 participates in the oxidation of pEGFP-Gn and pEGFP. The expression of EGFP gene in MCF7 is significantly increased in case of pEGFP-Gn. Stimulation of ROS synthesis (H2O2 40 μM or 10 cGy IR) increases the level of expression of EGFP. Conclusions GC-rich DNA fragments containing dGn motifs that are inclined to oxidation penetrate into MCF7 cancer cells, stimulate the adaptive response, and can be expressed. This property of GC-rich cell-free DNA should be considered and/or could potentially be used in therapy of tumors.

In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls—Part 2: Adaptive Response

“…The activation of the sensors launched synthesis of pro-inflammatory cytokines. By the example of model DNA fragments, which contained various numbers of GC-rich and oxidized motifs, it was previously shown that cfDNA could be a potential trigger of adaptive response in the cells and serve as an active agent in the realization of the bystander effect upon the exposure to ionizing radiation [ 22 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 56 , 57 , 58 ]. In this paper, we studied for the first time the ability of real cfDNA samples from human plasma to induce the adaptive response in the cells via the mechanism, which had been earlier described for model DNA fragments only.…”

“…It was found earlier that model samples of oxidized DNA (oxy-gDNA), in contrast to non-oxidized gDNA, induced an adaptive response in human cells, which is similar to the cells’ response to small doses of ionization radiation [ 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. The response includes the transient induction of reactive oxygen species (ROS) synthesis by NOX4 (NADPH-oxidase 4) pro-oxidant enzyme.…”

In Vitro Analysis of Biological Activity of Circulating Cell-Free DNA Isolated from Blood Plasma of Schizophrenic Patients and Healthy Controls

“…RIG-I functions as a cytosolic receptor detecting exogenous RNAs. RIG-I is able to detect 5′-triphosphorylated dsRNA transcribed from AT-rich dsDNA by DNA-dependent RNA polymerase III (Pol III) [ 42 , 59 , 60 , 61 ]. Apparently, cfDNA penetrating into the cell is transcribed with the formation of dsRNA molecules that interact with RIG-I.…”

Oxidative Modification of Plasmid Dna Promotes Their Penetration Into the Cytoplasma of Human Mesenchymal Stem Cells

“…Ранее мы показали, что окислительная модификация внеклеточной ДНК при добавлении в среду культивирования раковых клеток линии MCF7 способствует ее проникновению в цитоплазму клеток [3]. Облучение рентгеновским излучением в дозе 10 сГр раковых клеток MCF7 после добавления ГЦ-обогащённых плазмид в среду их культивирования приводило к повышению уровня 8-oxodG в плазмидах через 30 минут после облучения и к более интенсивному проникновению окисленных плазмид по сравнению с аналогичным экспериментом без облучения [4].…”