Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes

Abstract

Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored.We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results suggest that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.

Figures

Figure 1

Cellular entry, nuclear uptake and chromosomal association of fluorescently labelled DNAfs and Cfs. NIH3T3 cells (10×104) were treated with DNAfs labelled with ULS (red) and Cfs dual-labelled with ULS (red) and ATTO-TEC (green) (10 ng DNA in all experiments). (A) Intracellular fate of DNAfs at 30 min as analysed by LSCM. Numerous fine fluorescent particles are seen in the cytoplasm and in the nucleus. DIC, DAPI and ULS pictures are represented in different panels. (B) Intracellular fate of Cfs at 6 h as analysed by LSCM. Presence of dual-labelled Cfs in the cytoplasm and nuclei are clearly seen. The red and green signals appear yellow in colour when the images are overlapped. (C) Kinetics of nuclear uptake of fluorescently labelled DNAfs and Cfs analysed by LSCM. Fifty nuclei were analysed at each time-point and the percentage of positive nuclei was recorded. Nuclei containing at least two fluorescent spots were considered as positive. (D and E) Association of fluorescently labelled DNAfs (D) and Cfs (E) with chromosomes of treated cells. NIH3T3 cells were treated with labelled DNAfs and Cfs and metaphase spreads were prepared 6 h after treatment and analysed by fluorescence microscopy. Note that the labelled DNA particles are considerably smaller in size than Cfs particles.

Figure 2

Integration of DNAfs and Cfs into host cell genomes. (A) Representative FISH images showing presence of human genomic (red) and pan-centromeric (green) signals in mouse cell clone (B2) derived from NIH3T3 cells treated with Cfs. Upon merging the images, red and green signals frequently co-localize giving yellow fluorescence (marked by arrows). Summary results of FISH analysis depicted in a tabular form shows significantly higher frequency of human-specific signals (genomic + centromeric) in Cfs compared to DNAfs-derived clones. Fifty metaphases were counted in each case and the mean number of signals per metaphase was recorded and analysed by Student’s t-test. D5 vs E10 (p=0.0004); D5 vs E12 (p=0.0013); B2 vs E10 (p=0.0001); B2 vs E12 (p=0.0001). (B) Whole genome sequencing to detect presence of human sequence reads in DNAfs-derived and Cfs-derived clones. The histogram shows number of reads that are strictly human in nature in single-cell clones generated from DNAfs (E10, E12) and Cfs (B2, D5) treated NIH3T3 cells as well as in mouse reference genome (SRA ERP000354). The 2720 human sequence reads detected in the mouse reference genome are either an artifact of inefficiency of our alignment algorithm or an artifact of sequencing in the mouse reference genome. Control vs E10 (Chi-sq. 727.30; p<10–10); Control vs E12 (Chi-sq. 468.19; p<10–10); Control vs B2 (Chi-sq. 7502.97; p<10–10); Control vs D5 (Chi-sq. 4654.724; p<10–10); E10 vs B2 (Chi-sq. 4124.458; p<10–10); E10 vs D5 (Chi-sq. 1896.15; p<10–10); E12 vs B2 (Chi-sq. 4542.31; p<10–10); E12 vs D5 (Chi-sq. 2270.34; p<10–10). (C)In vivo FISH detection of human DNA in nuclei of vital organs of mice following intravenous injection of DNAfs and Cfs. Mice were intravenously injected with DNAfs and Cfs (100 ng DNA each) through tail vein and sacrificed on day 7. Control animals were injected with 100 μl of saline. Vital organs were removed and processed for FISH. The experiments were done in duplicate i.e., with two animals in each group. Two thousand cells were counted for each tissue per animal and the percentage of nuclei showing human-specific signals (genomic and/or centromeric) was calculated and analysed by Chi-square test. **p<0.01. The control animals did not show any human signals.

Figure 3

Activation of DNA damage and apoptotic pathways in response to DNAfs and Cfs derived from cancer patients. (A) Kinetics of H2AX phosphorylation in nucleic of NIH3T3 cells treated with DNAfs and Cfs. DNAfs and Cfs were isolated from pooled samples from cancer patients. NIH3T3 cells (10×104) were treated with DNAfs and Cfs (5 ng DNA each) and cells were processed for immuno-fluorescence to detect H2AX activation at various time points. Fifty nuclei in duplicate were analysed at each time-point and the percentage of nuclei showing positive signals was calculated. Nuclei with two or more fluorescent foci were considered as positive. (B) Analysis of various proteins involved in the DDR (upper panel) and apoptotic (lower panel) pathways induced by DNAfs and Cfs. NIH3T3 cells (10×104) were treated as described above and cells were processed for immuno-fluorescence at 6 h following treatment for detection of DDR proteins. Nuclei with two or more fluorescent foci were considered as positive. Experiments were done in duplicate; 50 cells were counted in each case and the percentage of positive cells was calculated and analysed by Chi-square test. For analysis of activation of apoptotic pathways, cells were treated as described above for 24 h and processed for detection of JC-1, Cytochrome-C and Caspase-3 by immuno-fluorescence. For JC-1, cell-associated fluorescence was detected after addition of JC-1 dye and the percentage of cells showing green fluorescence was calculated. For Cytochrome-C and Caspase-3, fluorescence was detected following antibody treatment and the percentage of positive cells was calculated. Experiments were done in duplicate, and 50 cells were examined in each case and analysed by Chi-square test. **p<0.01, ***p<0.001, ****p<0.0001. (C)In vivo activation of H2AX (upper panel) and active Caspase-3 (lower panel) in various organs and in PBMCs following intravenous injection of DNAfs and Cfs (100ng DNA each). Animals were sacrificed after 24 h, and various organs and PBMCs were processed for immuno-fluorescence. Control animals were injected with 100 μl of saline. The experiments were done in duplicate i.e., with two animals in each group. At least 1000 DAPI-stained nuclei per animal were examined from 10 randomly chosen areas of various tissues, and in case of PBMCs, 100 nuclei per animal were examined. In both cases, the number of nuclei showing positive foci (γ-H2AX) and number of cells showing positive fluorescence (Caspase-3) were recorded. The percentage of nuclei showing fluorescent foci (γ-H2AX) and percentage of cells showing positive nuclear fluorescence (active Caspase-3) were calculated and analysed by Chi-square test. *p<0.05, ****p<0.0001.

Figure 4

Induction of γH2AX and active Caspase-3 by DNAfs and Cfs derived from healthy volunteers. Samples from cancer patients were also analysed for comparison. DNAfs and Cfs were isolated from pooled plasma/serum of healthy volunteers and age- and sex-matched cancer patients. (A)In vitro analysis of γ-H2AX and active Caspase-3. NIH3T3 cells (10×104) were treated with DNAfs and Cfs (5 ng DNA each) for 6 h for detection of γ-H2AX (left) and for 24 h for detection of active Caspase -3 (right) by immuno-fluorescence. For γ-H2AX (left-hand panel), 300 nuclei were counted and the percentage of nuclei showing positive foci were calculated and analysed by Chi-square test. For active Caspase-3 (right-hand panel), 200 cells were counted and the percentage of cells showing positive fluorescent signals were calculated and analysed by Chi-square test. *p<0.05, **p<0.01, ****p<0.0001. (B)In vivo detection of γ-H2AX activation by DNAfs (upper panel) and Cfs (lower panel). Mice were injected intravenously with DNAfs and Cfs (100 ng DNA each) and vital organs were removed after 24 h and processed for γH2AX by immuno-fluorescence as described earlier. Control animals were injected with 100 μl of saline. The experiments were done in duplicate i.e., with two animals in each group. One thousand cells from each animal were analysed and the percentage of nuclei with positive fluorescent foci was calculated and analysed by Chi-square test. *p<0.05, **p<0.01, ****p<0.0001. (C)In vivo analysis of Caspase-3 activation by DNAfs (upper panel) and Cfs (lower panel). Mice were injected intravenously with DNAfs and Cfs (100 ng DNA each) and vital organs were removed after 24 h and processed for active Caspase-3 by immuno-fluorescence as described earlier. Control animals were injected with 100 μl of saline. The experiments were done in duplicate i.e., with two animals in each group; one thousand cells from each animal were analysed and the percentage of cells with positive fluorescence was calculated and analysed by Chi-square test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5

Genomic integration of Cfs involves DNA double-strand break repair. (A)In vitro demonstration of γ-H2AX foci at sites of Cfs integration. NIH3T3 cells (10×104) were treated for 6 h with Cfs (5 ng DNA) labelled in their protein with ATTO-TEC (green) and metaphase spreads were prepared. Immuno-fluorescence images were developed using antibody to γ-H2AX (red). Co-localization of green and red signals were clearly visible. (B)In vivo demonstration of γ-H2AX foci at the sites of Cfs integration. Mice were injected i.v. with Cfs (100 ng DNA) and sacrificed 24 h later. Sections of brain were processed for immuno-FISH using human-specific genomic probe (green) and antibody against γ-H2AX (red). Co-localization of green and red signals are clearly visible.

Figure 6

Schematic representation of proposed model of DNA damage. NHEJ = non-homologous end-joining; HR = homologous recombination; NHR = non-homologous recombination.