INTRODUCTION

Development of oncological diseases is often accompanied by the increase in the level of circulating cell-free DNA (cfDNA) in the blood of the patients [1]. There are three major sources of cfDNA: tumor cells, both located in the primary tumor node and circulating in the bloodstream at advanced stages of the disease; cells of the tumor microenvironment dying as the disease develops; and immune cells, mainly macrophages and neutrophils [2]. Composition of the circulating cfDNA depends on the type and stage of oncological disease; therefore, many researchers use analysis of the cfDNA profile for diagnosing and monitoring neoplastic process. For some types of cancer, where cfDNA composition largely reflects peculiarities of the DNA and chromatin of tumor cells, the genomic and epigenomic profiles are analyzed in the cfDNA pool, which makes it possible to estimate the tumor burden and to correct the treatment [3]. Analyses of the ratio of mutant fragments of oncogens, retrotransposons and mobile genetic elements (MGE), as well as of the changes in cfDNA methylation are successfully used in clinical and preclinical trials [4-6].

Although active attempts have been made to study cfDNA composition, not many studied have been performed on the functional role of cfDNA. The relatively well-studied objects are the so-called neutrophil extracellular traps (NETs) which are generated by neutrophils releasing their own genomic material and contents of the granules followed by formation of an extensive network, inter alia, in the tumor microenvironment [7]. NET formation in the tumor microenvironment provokes tumor growth and invasion [8]. Some types of tumor cells have been shown to have a CCDC25 receptor selectively recognizing precisely the DNA component of NET, 8-OH-deoxyguanosine (8-OhdG) residues, and activating migration of the tumor cells via the ILK-PARVB pathway [9].

Some researchers suggest that the circulating cfDNA is involved in horizontal transfer, penetrating into the healthy cells of a body causing their changes that lead to formation of the tumor microenvironment, pre-metastatic niche, and metastases [10]. In the field of study of the effects of tumor-specific cfDNA on the development of pathological processes, there are only a few pioneer works [11-13]. It has been shown that the tumor-specific cfDNA obtained from the patients with colorectal cancer is able to penetrate into the mouse fibroblasts [11]. It has been shown that cfDNA and chromatin from the blood serum of patients with different types of cancer are able, after penetrating into a cell, to enter the nucleus and to integrate into the genome of a recipient cell [13]. In their recent work, Souza et al. [14] have shown that cfDNA from the blood of patients with prostate cancer is absorbed by the nontumor prostate cell lines (RWPE-1 and PNT-2), which leads to the changes in the expression of some genes and miRNA associated with oncogenesis and intensification of cell migration properties. Our recent works show that the DNase I-induced decrease in concentration of the tumor-specific cfDNA in the blood serum of tumor-bearing mice with different metastatic forms of tumors correlated with the decrease in the primary tumor node and in the number of metastases, suggesting functional significance of cfDNA in the tumor progression [15-17]. It should be noted that cfDNA is a component of tumor microenvironment, which is constantly present at a high concentration and potentially affects both the tumor cells and the cells of the tumor microenvironment. However, there are still no studies describing the effect of cfDNA on tumor cells per se, in spite of the data on possibility of interaction between cfDNA and their receptors [9]. Thus, the study of cfDNA effects on tumor cells is a relevant issue and could be important for expanding our knowledge on the fundamental processes of oncogenesis.

The present work was aimed at studying biological effects of the cfDNA isolated from the blood serum of mice with metastatic melanoma B16 on the B16 cells in vitro. It has been shown that cfDNA is able to penetrate into the recipient cells and to activate expression of intracellular and secretory DNases in the latter, as well as Fos and Ras oncogenes triggering numerous signaling cascades, from apoptosis inhibition to increased tumor cell proliferation. Although cfDNA had no effect on viability and migration activity of the recipient cells, the cfDNA-induced activation of the expression of DNases and oncogenes could significantly contribute to enhancing oncogenic properties of tumor cells.

MATERIALS AND METHODS

Tumor cells. B16-F10 melanoma cells (hereinafter, B16 cells) were acquired from the Institute of Cytology of the Russian Academy of Sciences (St. Petersburg, Russia). B16 cells were incubated in a DMEM (Thermo Fisher Scientific, USA) supplemented with 10% fetal calf serum (Thermo Fisher Scientific), 1% antibiotic–antimycotic solution (100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin; Thermo Fisher Scientific) in 5% CO2 atmosphere at 37°C (standard conditions) and regular passages to maintain exponential growth.

Mice. Mice used in the work were 10-14-week-old male C57Bl/6 mice (hereinafter, C57Bl) bred at the vivarium of the Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (Novosibirsk, Russia). At the beginning of the experiment, the animal weight (mean ± SD) was 20.0 ± 1.5 g. The animals were kept in groups of 10 individuals per cage in a well-lighted room. The mice had free access to food and water.

Tumor model. A suspension of B16 cell (0.5×106 cells per ml) in 0.2 ml of a standard saline solution (0.9% aqueous solution of sodium chloride) was intravenously injected into mice to form a metastatic model of B16 melanoma. On day 21 after the injection of tumor cells, blood was taken intravitally from the retro-orbital sinus, and the mice were euthanized by cervical dislocation. Lungs were collected and fixed in a 10% formalin (pH 7.0; BioVitrum, Russia) for subsequent pathomorphological analysis. Number of surface metastases in the lungs were counted with a binocular microscope.

Obtaining a conditioned medium. B16 cells were incubated in a serum-free DMEM under standard conditions for 24 h. The medium was collected and used for cfDNA isolation.

Obtaining blood serum. Blood samples from mice (0.2 ml) were taken from the retro-orbital sinus. The serum from the blood samples of healthy mice and mice with metastatic melanoma B16 were obtained by clot formation at 37°C for 30 min and at 4°C overnight, followed by clot discard, and centrifugation (4000 rpm, 4°C, 20 min). Serum samples were stored at –70°C.

cfDNA isolation. cfDNA from the conditioned medium (B16CM-cfDNA), the serum samples of healthy mice (h-cfDNA), and mice with metastatic melanoma B16 (B16S-cfDNA) was isolated by phenol/chloroform (1/1; v/v) extraction followed by reversible sorption on a silica-based sorbent with a QI Aquick Gel Extraction kit (Qiagen, USA) [17]. Purity of the isolated DNA was assessed using a NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific); concentration was measured with a Qubit™ fluorimeter (Invitrogen, USA) and a Quant-iT™ dsDNA HS Assay kit (Thermo Fisher Scientific).

Total DNA isolation from B16 cells (B16-DNA). Medium was removed from B16 cells; the cells were detached with a 0.25% trypsin solution in EDTA, washed with PBS, and pelleted by centrifugation at 300g for 5 min. The cell pellet was treated with a 0.25% trypsin solution in EDTA and centrifuged at 350g for 20 min at 4°C, followed by removal of the supernatant. Next, the cells were lysed in 600 µl of a solution containing 100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 200 mM NaCl, 0.2% SDS, and 10 ng/ml proteinase K for 4 h at 65°C with constant mixing. After that DNA was extracted with a phenol/chloroform (1/1; v/v) mixture as described above and precipitated with 96% ethanol in the presence of 0.3 M sodium acetate at ‒20°C for 18 h. DNA was isolated by centrifugation; the pellet was washed with 80% ethanol, dried out, dissolved in water, and stored at –20°C. DNA concentration was determined with a Qubit™ fluorimeter and a Quant-iT™ dsDNA HS Assay kit.

Study of the effect of cfDNA on B16 melanoma cells. B16 cells were seeded into a serum-free DMEM to a 24-well microplate at a density of 0.5×106 cells per well until reaching a 95% confluency in 12 h. In 24 h, the medium was replaced with a fresh serum-free DMEM, h-cfDNA or B16S-cf-DNA were added to a concentration of 100 ng/ml, followed by incubation under standard conditions for 24 h. The cells were harvested. One part of the cell pellet was used for phenol/chloroform extraction of total DNA, followed by its precipitation with ethanol as described above. The second part of the cell pellet was used for RNA isolation using Trizol (Thermo Fisher Scientific) according to the standard protocol. Quality and concentration of isolated RNA were assessed using a NanoDrop® ND-1000 spectrophotometer; DNA quality and concentration were estimated with a Qubit™ fluorimeter and a Quant-iT™ dsDNA HS Assay kit.

Determination of the number (abundance) of MGEs and oncogene fragments in cfDNA using qPCR. Amount of MGE and oncogene sequences within B16-DNA, B16CM-cfDNA, h-cfDNA, and B16S-cfDNA were determined by qPCR. Amplification was performed in 20 µl of reaction mixture containing 0.1-0.5 ng of DNA, SYBR-Green-containing Bio Master CorHS-qPCR (Biolabmix, Russia), and 0.6 µM of each forward and reverse specific primers for MGE (B1_mus2, L1td1) and oncogenes (Braf, Hmga2, Myc, Fos, Jun, Ras, P53) (Table 1). Reaction conditions were as follows: 95°C, 6 min; 95°C, 15 s; 60°C, 20 s; 70°C, 60 s, 50 cycles. Gapdh was used as reference gene.

Table 1 Sequences of primers used in the work
Full size table

Determination of the level of expression of MGE, DNases, and oncogenes using RT-qPCR. cDNA was prepared in 40 µl of reaction mixture containing 2 µg of total cellular RNA, 5× RT buffer mix (Biolabmix), 100 U of reverse transcriptase MMuLV-RH (Biolabmix), and 0.05 µM of primer dT15. Reverse transcription reaction was carried out in the following mode: 42°C, 1 h; 70°C, 10 min.

Reaction mixture for qPCR (25 µl) contained: 0.1-0.5 ng of cDNA, BioMaster HS-qPCR SYBR Blue (Biolabmix), and 0.6 µM of each forward and reverse specific primers for MGEs (L1td1 Orf1, L1td1 Orf2), DNases (Dnase1l3, Dffa, Dffb, Endog), and oncogenes (Fos, Jun, Myc, Ras) (Table 1). Reaction conditions were as follows: 95°C, 6 min; 95°C, 15 s; 60°C, 20 s; 70°C, 60 s, 45 cycles. β-actin was used as reference.

Study of cfDNA effect on viability, migration activity, and apoptosis of B16 cells. MTT test. Cells were seeded in a 96-well microplate, 1.5×104 cells per well, in a serum-free DMEM. After 24 h, the medium was replaced with a fresh serum-free DMEM, with addition of h-cfDNA or B16S-cfDNA at concentrations of 5-100 ng/ml, and cells were incubated under standard conditions for 24 h. Next an MTT solution was added to the cells to a concentration of 0.5 mg/ml, incubated for 3 h, and analyzed as described previously [18].

Scratch test. The cells in a serum-free DMEM were seeded into a 6-well microplate, 1.5×106 cells per well, until reaching 90% confluency in 12 h. 0.5-mm wide scratches were made in the cell monolayer with a tip of a 200-µl pipette; the monolayer was washed three times with PBS, followed by addition of a serum-free DMEM, h-cfDNA or B16S-cfDNA to a final concentration of 100 ng/ml, and cells were incubated under standard conditions for 24 h. Boundaries of a scratch were marked, and damaged zones were photographed periodically over 24 h using a Zeiss Primo Vert microscope (Zeiss, Germany). The rate of cell migration was estimated with ImageJ [17].

Apoptosis. B16 cells in a serum-free DMEM were seeded into a 24-well microplate, 0.5×106 cells per well. After 12 h, the medium was replaced with a fresh serum-free DMEM, followed by addition of h-cfDNA and B16S-cfDNA to a final concentration of 100 ng/ml, cells were incubated under standard conditions for 24 h. Apoptosis was analyzed with an Annexin V-FITC kit (Abcam, Great Britain) according to the manufacturer’s protocol using a NovoCyte flow cytometer (ACEA Biosciences, USA).

B16 cells incubated in a serum-free DMEM for 24 h were used as a control for the MTT test, Scratch test, and apoptosis measurement.

Statistical analysis. Data were processed using Student’s t-test (two tailed, unpaired) and one-way analysis of variance (ANOVA) with a post hoc Tukey test. The value p ≤ 0.05 was considered statistically significant. Statistical analysis was performed using STATISTICA 10.0.

RESULTS

Obtaining and characterization of cfDNA samples from blood serum. Melanoma B16 cells were injected intravenously into C57Bl mice (105 cells/mouse); after 21 days, blood was taken, the mice were euthanized, and the number of surface lung metastases was determined. cfDNA was isolated from the blood serum as described in “Materials and Methods” section. The scheme for obtaining cfDNA samples is presented in Fig. 1a. In the case of intravenous injection, B16 melanoma develops as numerous metastatic nodes, primarily in the pulmonary tissue of animals (Fig. 1b). On the day 21 of tumor development, the number of surface metastases was 50 on average; however, there was a dispersion in the number of metastases per individual: from 25 to 150 (Fig. 1c).

Fig. 1.
figure 1

Characterization of the metastatic melanoma model. a) Experimental design. b) Lungs of healthy mice and tumor–bearing mice with the number of surface metastases <50 and >50. c) Dispersion of metastases number in tumor-bearing mice <50 (n = 20) and >50 (n = 35). d) cfDNA concentration in the blood serum of healthy and tumor–bearing mice with the number of metastases >50 (n = 35). The data were analyzed using one-way ANOVA with a post hoc Tukey test and are presented as a median, ** p < 0.001.

Full size image

Tumor development (metastases in the lungs) in mice was accompanied by a considerable increase in the circulating cfDNA concentration in the blood serum of tumor-bearing mice (Fig. 1d). For example, the median value of cfDNA concentration in the blood serum of tumor-bearing mice was 1000 ng/ml, while the values varied from 100 to 2500 ng/ml (Fig. 1d). The median value of cfDNA concentration in the blood serum of healthy mice was 35 ng/ml and the values varied from 0 to 150 ng/ml (Fig. 1d).

Further experiments were performed using samples of blood serum from the tumor-bearing mice with no less than 25 surface lung metastases and cfDNA concentration in the serum of no less than 250 ng/ml, as well as samples of blood serum from the healthy mice with cfDNA concentration of no less than 50 ng/ml. The isolated cfDNAs were combined by groups. Thus, the combined cfDNA preparations of tumor-bearing animals (B16S-cfDNA) and healthy animals (h-cfDNA) were obtained. In addition, cfDNA from the conditioned medium of B16 cells (B16CM-cfDNA) and total DNA of B16 cells (B16-DNA) were isolated to compare fragment compositions.

cfDNA preparations were characterized with respect to the abundance for the oncogene and MGE fragments by qPCR. Table 2 shows the data on abundance of B16CM-cfDNA, h-cfDNA, and B16S-cfDNA for MGE and oncogene fragments. The abundance of MGE and oncogene fragments in the B16-DNA was used as a reference.

Table 2 Abundance of cfDNA from blood serum of the healthy mice and the mice with melanoma, genomic DNA of B16 cells, and conditioned medium of B16 cells for MGE and oncogene fragments
Full size table

One can see that the sequences of MGE and oncogenes are weakly represented in the h-cfDNA compared to their abundance in the B16-DNA. Compositions of B16S-cdDNA and B16CM-cfDNA were similar in terms of the abundance of the selected fragments: the Hmga2, Braf, P53, and Jun gene sequences were detected at low levels; the B1_mus1, B1_mus2, L1td1 Orf2, and Myc sequences were represented at a rather high level: more than 0.7 a.u. (Table 2). The differences were found only for the Ras gene, with higher abundance of its fragments in the conditioned medium compared to both B16-DNA and B16S-cfDNA.

Study of the effect of B16S-cfDNA on the properties of B16 melanoma cells. The effect of B16S-cfDNA on the properties of B16 cells was studied by adding h-cfDNA or B16S-cfDNA to the cells in the serum-free DMEM to final concentration of 100 ng/ml, followed by incubation under standard conditions for 24 h. After the incubation, the cells were harvested, total DNA was isolated, and abundance of the fragments of oncogenes (Myc, Ras) and MGE (B1_mus2, L1td1) was determined by qPCR (Fig. 2a).

Fig. 2.
figure 2

Effect of B16S-cfDNA on B6 melanoma cells. a) Experimental scheme. b) Change in the abundance of the Myc, Ras, L1td1, and B1_mus2 fragments in the B16 cells after incubation with the B16S-cfDNA. The data of qPCR. The reference gene was Gapdh. c-e) Effect of B16S-cfDNA on the levels of expression of DNases (c), MGEs (d) and oncogenes (e) in the B16 cells. The data of RT-qPCR. The reference gene was β-actin. Control: intact cells. The data were analyzed using Student’s t-test and presented as the mean ±SEM; * p < 0.05; ** p < 0.001.

Full size image

The above data (Fig. 2b) show that the B16S-cfDNA leads to a multifold increase in the abundance of the fragments of both oncogenes and B1_mus2. H-cfDNA caused a reliable increase in the abundance of only B1_mus2 fragments in the B16 melanoma cells and had no effect on the abundance of the oncogenes and L1td1 fragments (Fig. 2b). Neither h-cfDNA nor B16S-cfDNA caused reliable increase in the abundance of the L1td1 fragments in B16 cells. Thus, it can be concluded that cfDNA is able to penetrate into the B16 melanoma cells.

In addition to the changes in the abundance of the oncogene and MGE fragments, the changes in the recipient cells after their incubation with B16S-cfDNA were assessed by the changes in the mRNA levels of a number of key DNases (Dffa, Dffb, Dnase1L3, EndoG), which are markers of the primary cell response to the presence of exogenous DNA, oncogenes (Ras, Myc, Fos, Jun) triggering intracellular processes and being the main indicators of cell metabolism, and the LINE element of L1td1 (Orf1, Orf2) indicating activation/suppression of retrotransposition (Fig. 2c).

It has been shown that incubation with h-cfDNA results in 2-fold increase of the mRNA level of Dnase1L3 encoding intracellular secretory DNase and causes 5-fold increase in the mRNA level of EndoG encoding the DNase involved mainly in the negative regulation of apoptosis and limiting expression of another secretory DNase – DNase I (Fig. 2c). At the same time, the level of Dffb mRNA expression encoding the apoptotic DNase was unchanged. Effect of the B16S-cfDNA on B16 melanoma cells was considerably different from the effect of h-cfDNA: incubation with the B16S-cfDNA results in the significant increase in the level of Dnase1L3 mRNA expression compared to the intact cells (the control) and a statistically insignificant increase in the expression of mRNA of Dffb and EndoG (Fig. 2c). Interestingly, incubation with the cfDNA of both types led to the reliable decrease in the level of Dffa mRNA expression.

Incubation with both B16S-cfDNA and h-cfDNA increased expression of the L1td1 (Orf1 and Orf2) mRNA; however, the effect of h-cfDNA was more pronounced (Fig. 2d). It should be noted that h-cfDNA caused more marked changes in the gene expression in B16 melanoma cells than B16S-cfDNA: h-cfDNA caused a 4-fold increase in the mRNA level of Fos and Ras oncogenes compared to the control (Fig. 2e), while B16S-cfDNA caused a weak, statistically insignificant increase in the expression of all four oncogenes under study (Fig. 2e).

One of the objectives of the present research was to study how the cfDNA of tumor origin affects viability and migration activity of the tumor cells and whether cfDNA is able, after penetrating into tumor cells, to trigger their apoptosis in vitro. The level of apoptosis was determined as follows: B16 melanoma cells were incubated in the presence of B16S-cfDNA at a concentration of 100 ng/ml for 24 h, then stained with annexin V-FITC/PI and analyzed by flow cytometry (Fig. 3a). Viability of the B16 cells was determined by the MTT test after their incubation in the presence of h-cfDNA or B16S-cfDNA at concentration of 10-100 ng/ml for 24 h (Fig. 3b). The effect of cfDNA on B16 cell migration was studied by the scratch test: B16 cells were incubated in the presence of h-cfDNA or B16S-cfDNA at a concentration of 100 ng/ml to test the rate of overgrowth of a scratch formed in the monolayer (Fig. 3, c and d).

Fig. 3.
figure 3

Effect of B16S-cfDNA on apoptosis, viability, and migration activity of B16 melanoma cells. a) Effect of cfDNA on apoptosis of B16 cells. The results of flow cytometry. Q3-1, Annexin V-FITC–/PI+, necrosis; Q3-2, annexin V-FITC+/PI+, late apoptosis; Q3-3, annexin V-FITC–/PI–, live cells; Q3-4, annexin V-FITC+/PI–, early apoptosis. Positive control of late apoptosis: B16 cells treated with staurosporine (0.1 µg/mln cells). b) Effect of cfDNA on viability of B16 cells. The data of the MTT test. The data are presented as the mean ± SEM. c) Degree of scratch healing by B16 cells after 24-h incubation with cfDNA. The data of the Scratch test. d) Photographs of scratch healing by B16 cells after 24-h incubation with cfDNA (4-fold magnification). Solid line: scratch boundaries at the moment of time 0; dotted lines: the cell front boundary in 24 h. Control: intact untreated B16 cells.

Full size image

It has been shown that neither h-cfDNA nor B16S-cfDNA affect the level of apoptosis, viability, and migration properties of B16 cells (Fig. 3). Thus, the autologous cfDNA did not change macro properties of the melanoma cells, either in terms of enhancing of pro-tumor activity (proliferation, migration) or in terms of triggering of antitumor activity (apoptosis).

DISCUSSION

At present, there is a large amount of data on composition of the circulating cfDNA in the blood of higher organisms with different pathologies, but biological functions of cfDNA are still being actively studied. In clinical practice, attempts have been made to use characteristics of cfDNA for early diagnosis of cancer diseases [2]. The comprehensive study of cfDNA characteristics made it possible to pose a question about the potential role of cfDNA in carcinogenesis, which has been confirmed by some experimental data [11, 13, 14, 19]. In a few works it has been suggested that the tumor-associated cfDNA comprising, inter alia, fragments of oncogenes, could behave similar to oncoviruses, which opens up an alternative pathway to metastasis [11, 13, 14]. Discovery of the DNA-containing microvesicles and evidence of horizontal DNA transfer for many cell lines and organisms strengthened this hypothesis [19]. In spite of fact that some data on the functional effect of the tumor-derived cfDNA on healthy cells were reported, there are no data on the effect of such cfDNA on tumor cells.

The aim of this work was to study the effects of circulating B16S-cfDNA on tumor cells in order to understand mechanisms mediating involvement of the circulating tumor-specific cfDNA in oncotranformation. The tumor-associated cfDNA used in our work was the B16S-cfDNA from blood serum of the mice with metastatic melanoma B16 characterized by the large number of metastases in lungs and high concentration of cfDNA in the blood serum. Taking into account that the tumor cells are also exposed to the pressure of cfDNA generated by the healthy cells, especially at the early stages of tumor development, h-cfDNA was also used in the experiments.

We have shown that the composition of B16S-cfDNA is significantly different from the composition of h-cfDNA and its abundance with oncogene and MGE sequences is similar to those of both total B16-DNA and B16CM-cfDNA. These results confirm that cfDNA in the case of metastatic melanoma is mainly tumor-associated and represents composition of the genomic DNA of B16 cells, which makes it possible to consider this cfDNA as tumor-specific. This is in agreement with the fact that the genome of tumor cells differs from the genome of normal cells and, in many cases, the modified profile of cfDNA shows that the DNAs from tumor cells in this pool are prevalent over the DNAs from normal cells [20].

It is interesting that neither B16S-cfDNA nor h-cfDNA had an effect on pro- and anti-tumor activity. Mitra et al. [13] have shown that cfDNA from the blood of cancer patients could penetrate into the cell nuclei and be adhered by chromatin, which resulted in the double-strand breaks in DNA and, as a consequence, triggered early stages of apoptosis in the recipient cells. Nevertheless, it should be noted that Mitra et al. [13] used human cfDNA and NIH3T3 mouse fibroblasts as recipient cells; therefore, induction of apoptosis could be explained by a species conflict [21, 22]. Both the present work and the work by Mitra et al. [13] were performed with the same cfDNA concentrations (100 ng/ml). Nevertheless, our data show that cfDNA concentrations in the blood of tumor-carrying animals can reach 1500 ng/ml and, hence, the continuously replenished pool of tumor-specific cfDNA at high concentrations under in vivo conditions could induce more pronounced apoptotic signals accompanied by the strengthening or weakening of invasive properties of tumor cells.

Nevertheless, in spite of the absence of cfDNA effects on viability, apoptosis, and migration of the B16 melanoma cells, it has been shown that both B16S-cfDNA and h-cfDNA cause changes in the mRNA levels of some of the key genes: DNases, as primary response genes induced in response to exogenous DNA; oncogenes that trigger intracellular cascades, including the cascades mediating oncotransformation [23]; and L1td1 retrotransposon.

It has been shown that neither type of cfDNA induced increase in the level of expression of Dffb mRNA encoding the pro-apoptotic DNase, which may indicate absence of toxic effects of cfDNA. Nevertheless, both types of cfDNA induced decrease in the level of the Dffa DNase mRNA expression, which is a specific inhibitor of the Dffb DNase. Normally, both proteins are inactive heterodimers and, in the case of an external threat such as foreign DNA or increase in the total concentration of intracellular DNA, there is dissociation of the heterodimer with release of the active subunit of Dffb [24]. A new portion of Dffa is required to suppress the excessive activity of Dffb; however, the reduced expression of the Dffa mRNA encoding the inhibitor protein suggests higher level of the cell readiness to DNA cleavage [25].

B16S-cfDNA caused a very moderate increase in the mRNA level of the gene encoding EndoG, a broad-spectrum nuclease, negative regulator of apoptosis involved in the repair processes [26]. Under normal conditions, EndoG is localized in mitochondria; only when mitochondria are destroyed during apoptosis, it enters the nucleus, where it could cause chromatin destruction, activation of other DNases, and intensification of the apoptotic cascade [27]. Our data indicate that the increased level of the EndoG mRNA could correlate with the limited triggering of apoptosis. It should be noted that the level of EndoG mRNA increased to a much greater extent in response to h-cfDNA than in response to B16S-cfDNA.

B16S-cfDNA caused a significant increase in the level of Dnase1l3 mRNA encoding the secreted DNase. The Dnase1l3 is able to cleave DNA in the DNA–protein complexes and is a powerful tool for protecting cells against foreign DNA or their own DNA, when its concentration increases as a result of metabolic processes in an organism [28, 29]. Thus, cell incubation in the presence of cfDNA switches on the mechanisms of cell defense against excessive DNA.

Analysis of the effect of B16S-cfDNA on the levels of oncogenes in the B16 cells showed only a minor decrease in the mRNA level of oncogenes Fos, Jun (encodes transcription regulators) and Ras (encodes the messenger protein involved in many signaling cascades). At the same time, h-cfDNA caused a considerable (4-fold) increase in the mRNA level of the Fos and Ras oncogenes relative to the control, while the expression of Jun did not increase [30].

The Jun and Fos proteins are prone to dimerization and normally there are either Jun-Jun and Fos-Fos dimers with a relatively low DNA-binding ability [31], or mixed Jun-Fos dimers, which are more stable and have a stronger DNA-binding ability compared to the homodimers [31]. The observed increase in the mRNA level of Fos is the evidence of activation of the cell signaling cascades, which can stimulate proliferation and invasion of the tumor cells. The observed increase in the mRNA level of Ras (the gene of the small GTPase, which is an intermediate link in the signal transduction chain in many cell cascades) is also an indicator of activation of the cell signaling cascades leading to enhanced oncotransformation [32].

The revealed ability of h-cfDNA to induce activation of the signaling pathways in tumor cells at the mRNA level, which intensify oncotransformation to a much greater extent than B16S-cfDNA, is an interesting result. The tumor-specific circulating cfDNA differs from the conditionally normal circulating cfDNA by the changed methylation status and can activate signaling pathways via the intracellular TLR9 receptors usually recognizing nonmethylated CpG motifs [33]. In addition, GC composition of the tumor-specific cfDNA, as a rule, differs from that of the conditionally normal circulating cfDNA, by an increase in the abundance of GC-rich sequences, which can also lead to the changes in TLR9 signaling [34]. The tumor-specific cfDNA can be characterized by the increased abundance of 8-oxodG, which affects its recognition by receptors and, accordingly, on the signaling pathways it triggers in the cells [35]. Thus, the levels of activation of intracellular signaling in the tumor cells in response to tumor-specific DNA, which is autologous to B16 cells, and conditionally normal DNA are different.

CONCLUSIONS

Summarizing all the above, it can be concluded that both cfDNA from the blood serum of healthy mice and cfDNA from the blood serum of mice with metastatic melanoma B16 induce a marked response in the B16 cells: the enhanced expression of intracellular and secreted DNases, which not only protect the cells from the excessive DNA concentrations but also regulate apoptotic processes. The cfDNA of healthy mice increased the mRNA level of the Fos and Ras oncogenes triggering a large number of signaling cascades, from apoptosis inhibition to increased tumor cell proliferation. Activation of transcription and intracellular signaling processes in the tumor cells in response to cfDNA of both types, which has been demonstrated in vitro, can increase susceptibility of tumor cells to other stimuli (histones, low-molecular mediators, growth factors, etc.) circulating in the bloodstream together with cfDNA in vivo [36]. Eventually, total mobilization of the cellular processes can contribute to tumor cell proliferation, formation of tumor microenvironment, increased tumor node, increased migration and, finally, more intensive oncogenesis, formation of a premetastatic niche and metastasis.