Illegitimate and Repeated Genomic Integration of Cell-Free Chromatin in the Aetiology of Somatic Mosaicism, Ageing, Chronic Diseases and Cancer

Abstract

Emerging evidence suggests that an individual is a complex mosaic of genetically divergent cells. Post-zygotic genomes of the same individual can differ from one another in the form of single nucleotide variations, copy number variations, insertions, deletions, inversions, translocations, other structural and chromosomal variations and footprints of transposable elements. High-throughput sequencing has led to increasing detection of mosaicism in healthy individuals which is related to ageing, neuro-degenerative disorders, diabetes mellitus, cardiovascular diseases and cancer. These age-related disorders are also known to be associated with significant increase in DNA damage and inflammation. Herein, we discuss a newly described phenomenon wherein the genome is under constant assault by illegitimate integration of cell-free chromatin (cfCh) particles that are released from the billions of cells that die in the body every day. We propose that such repeated genomic integration of cfCh followed by dsDNA breaks and repair by non-homologous-end-joining as well as physical damage to chromosomes occurring throughout life may lead to somatic/chromosomal mosaicism which would increase with age. We also discuss the recent finding that genomic integration of cfCh and the accompanying DNA damage is associated with marked activation of inflammatory cytokines. Thus, the triple pathologies of somatic mosaicism, DNA/chromosomal damage and inflammation brought about by a common mechanism of genomic integration of cfCh may help to provide an unifying model for the understanding of aetiologies of the inter-related conditions of ageing, degenerative disorders and cancer.

Keywords: DNA damage; NFκB; apoptosis; cell death; chromosomal damage; chromosomal mosaicism; chromosomes; circulating nucleic acids; inflammation.

Figure 1

Levels of cell-free chromatin (cfCh) in serum increase with age. The study included 140 healthy subjects aged 15–70 years. The cfCh levels were measured using the Cell Death Detection ELISAPlus kit (Roche Apllied Sciences, Mannheim, Germany). Values are expressed as arbitrary units (AU). Reproduced from [62].

Figure 2

Electron microscopy image of cfCh isolated from pooled sera of cancer patients showing beads-on-a-string appearance typical of chromatin of disparate sizes. Reproduced from [64].

Figure 3

Genomic integration of cfCh in NIH3T3 cells leads to DNA double-strand breaks. NIH3T3 cells were treated with cfCh isolated from sera of cancer patients, labelled in their histones with ATTO 488 NHS-ester (ATTO-TEC GmbH, Germany. Catalogue No. AD 488-35) and applied to NIH3T3 cells. After several passages, metaphase spreads were prepared and immune-stained with antibody to γH2AX. Co-localization of green (cfCh) and red (γH2AX) signals are clearly seen under florescent microscopy. Magnification x60. Reproduced from [64].

Figure 4

Genomic integration of cfCh in brain cells involves DNA double-strand break repair. Mice were injected intravenously with cfCh (100 ng DNA) isolated from cancer patients and sacrificed 24 h later. Sections of brain were processed for immuno-FISH using a human-specific whole genomic probe (green) and antibody against γ-H2AX (red). Co-localization of green and red signals are clearly visible. Magnification x60. Reproduced from [64].

Figure 5

Confocal microscopy images to demonstrate bystander uptake by NIH3T3 cells of dually labeled cfCh particles released from irradiated dying Jurkat cells that had been labeled in their DNA with BrdU (red) and histones by CellLight^® Histone 2B-GFP (green). (A). dually labeled Jurkat cells. (B). NIH3T3 cells that had been co-cultivated with irradiated (15 Gy) dually labeled Jurkat cells at 24 h. Reproduced from [84].

Figure 6

Detection of numerous fluorescent cfCh signals in nuclei of brain cells of mice following intravenous injection of fluorescently dually labelled MDA-MB-231 human breast cancer cells. MDA-MB-231 cells were dually labelled in their DNA with BrdU and in their histone H2B with CellLight^® Histone 2B GFP as described in [84]. One hundred thousand cells were injected intravenously, and animals were sacrificed after 72 h; sections of brain were examined by fluorescent microscopy as described in reference [64]. Magnification x60. (Unpublished data from authors’ lab).

Figure 7

Co-localization of BrdU labelled fluorescent cfCh signals with those of γH2AX and NFκB in nuclei of cells of vital organs of mice. BrdU pre-labelled B16-F10 mouse melanoma cells were treated with Adriamycin and 10 × 10⁴ dying cells were injected intravenously. Animals were sacrificed after 72 h, vital organs were immuno-stained with antibodies against γH2AX and NFκB and examined by fluorescence microcopy. Magnification x40. Reproduced from [83].

Figure 8

Schematic representation of a proposed model of DNA damage and inflammation following cellular uptake of cfCh. NHEJ = non-homologous end-joining; HR = homologous recombination; NHR = non-homologous recombination. Reproduced with modification from Chaudhary et al. 2018.

Figure 9

FISH detection of genomic integration of human DNA in mouse cells highlighting co-localization of human genomic and human pan-centromeric signals (arrow heads). Metaphase spreads were prepared from a single cell clone developed from NIH3T3 mouse fibroblast cells treated with cfCh isolated from sera of cancer patients [64]. FISH was performed using human whole genomic and human pan-centromeric probes as described in reference [64]. Magnification x60. (Unpublished data form authors’ lab).

Figure 10

Chromosomal aberrations induced in healthy cells by cfCh released from dying cancer cells. NIH3T3 mouse fibroblast cells were grown in conditioned medium of irradiated (10 Gy) MDA-MB-321 human breast cancer cells for 96 h. Cell culture was continued in fresh medium and karyotype analysis was performed at the 10th passage. (A,B) wagon-wheel representation of chromosomal aberrations in control and conditioned medium treated NIH3T3 cells, respectively. Fifteen metaphases were analyzed in each case and average percentage values are given in the figures. (C) depiction of various chromosomal abnormalities. Reproduced from [84].

Figure 11

Co-localization of γH2AX and NFκB fluorescent signals in vital organs of mice following intravenous injection of Adriamycin treated dying B16-F10 cells. One hundred thousand cells were injected intravenously into mice and animals were killed after 72 h. The vital organs were removed and stained with antibodies against γH2AX and NFκB and examined by fluorescence microscopy. Magnification x40. Reproduced from [83].

Figure 12

Schematic representation of a proposed model to explain detection of co-localized fluorescent signals of γH2AX and NFκB. Reproduced with modification from [96].

Figure 13

Positive correlation between serum cfCh levels and inflammatory cytokines (IL-6 and TNF-α). The study included 140 healthy subjects aged 15–70 years. cfCh levels were measured using the Cell Death Detection ELISAPlus kit (Roche Applied Sciences, Mannheim, Germany). Results are expressed in arbitrary units (AU). IL-6 and TNF-α were measured using cytometric bead array assay kit (BD Biosciences, San Jose, CA, USA). Reproduced from [62].