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

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

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

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

Imbalance of mitochondrial-nuclear cross talk in isocyanate mediated pulmonary endothelial cell dysfunction

Mechanistic investigations coupled with epidemiology, case-control, cohort and observational studies have increasingly linked isocyanate exposure (both chronic and acute) with pulmonary morbidity and mortality. Though ascribed for impairment in endothelial cell function, molecular mechanisms of these significant adverse pulmonary outcomes remains poorly understood. As preliminary studies conducted in past have failed to demonstrate a cause-effect relationship between isocyanate toxicity and compromised pulmonary endothelial cell function, we hypothesized that direct exposure to isocyanate may disrupt endothelial structural lining, resulting in cellular damage. Based on this premise, we comprehensively evaluated the molecular repercussions of methyl isocyanate (MIC) exposure on human pulmonary arterial endothelial cells (HPAE-26). We examined MIC-induced mitochondrial oxidative stress, pro-inflammatory cytokine response, oxidative DNA damage response and apoptotic index. Our results demonstrate that exposure to MIC, augment mitochondrial reactive oxygen species production, depletion in antioxidant defense enzymes, elevated pro-inflammatory cytokine response and induced endothelial cell apoptosis via affecting the balance of mitochondrial-nuclear cross talk. We herein delineate the first and direct molecular cascade of isocyanate-induced pulmonary endothelial cell dysfunction. The results of our study might portray a connective link between associated respiratory morbidities with isocyanate exposure, and indeed facilitate to discern the exposure-phenotype relationship in observed deficits of pulmonary endothelial cell function. Further, understanding of inter- and intra-cellular signaling pathways involved in isocyanate-induced endothelial damage would not only aid in biomarker identification but also provide potential new avenues to target specific therapeutic interventions

Cell-free chromatin particles released from dying host cells are global instigators of endotoxin sepsis in mice.

We have earlier reported that cell-free chromatin (cfCh) particles that are released from dying cells, or those that circulate blood, can readily enter into healthy cells, illegitimately integrate into their genomes and induce dsDNA breaks, apoptosis and intense activation of inflammatory cytokines. We hypothesized that sepsis is caused by cfCh released from dying host cells following microbial infection leading to bystander host cell apoptosis and inflammation which are perpetuated in a vicious cycle with release of more cfCh from dying host cells. To test this hypothesis we used three cfCh inactivating agents namely 1) anti-histone antibody complexed nanoparticles which inactivate cfCh by binding to histones; 2) DNase I which inactivates cfCh by degrading its DNA component, and 3) a novel pro-oxidant combination of Resveratrol and Copper which, like DNase I, inactivates cfCh by degrading its DNA component. Female C57 BL/6 mice, 6-8 weeks old, were administered a single i.p. injection of LPS at a dose of 10 mg/Kg or 20 mg/Kg with or without concurrent treatment with the above cfCh inactivating agents. Administration of cfCh inactivating agents concurrently with LPS resulted in prevention of following pathological parameters: 1) release of cfCh in extra-cellular spaces of brain, lung and heart and in circulation; 2) release of inflammatory cytokines in circulation; 3) activation of DNA damage, apoptosis and inflammation in cells of thymus, spleen and in PBMCs; 4) DNA damage, apoptosis and inflammation in cells of lung, liver, heart, brain, kidney and small intestine; 5) liver and kidney dysfunction and elevation of serum lactate; 6) coagulopathy, fibrinolysis and thrombocytopenia; 7) lethality. We conclude that cfCh that are released from dying host cells in response to bacterial endotoxin represents a global instigator of sepsis. cfCh inactivation may provide a novel approach to management of sepsis in humans

Source of FISH probes and antibodies.

Metastatic dissemination following successful treatment of the primary tumour remains a common cause of death. There is mounting evidence that therapeutic interventions themselves may promote development of metastatic disease. We earlier reported that cell-free chromatin particles (cfChPs) released from dying cancer cells are potentially oncogenic. Based on this observation we hypothesized that therapeutic interventions may lead to the release of cfChPs from therapy induced dying cancer cells which could be carried via the blood stream to distant organs to transform healthy cells into new cancers that would masquerade as metastasis. To test this hypothesis, we generated xenografts of MDA-MB-231 human breast cancer cells in severe combined immune-deficient mice, and using immuno-fluorescence and FISH analysis looked for cfChPs in their brain cells. We detected multiple human DNA signals representing cfChPs in nuclei of brain cells of mice which co-localized with eight human onco-proteins. No intact MDA-MB-231 cells were detected. The number of co-localizing human DNA and human c-Myc signals increased dramatically following treatment with chemotherapy, localized radiotherapy or surgery, which could be prevented by concurrent treatment with three different cfChPs deactivating agents. These results suggest that therapeutic interventions lead to the release cfChPs from therapy induced dying cancer cells carrying oncogenes and are transported via the blood stream to brain cells to potentially transform them to generate new cancers that would appear as metastases. cfChPs induced metastatic spread of cancer is preventable by concurrent treatment with agents that deactivate cfChPs.</div

Quantitative analyses depicted as histograms to illustrate that therapeutic interventions promote systemic dissemination of human DNA and human c-Myc oncoprotein to mouse brain cells, and that these can be prevented by concurrent treatment with three different cfChPs deactivating agents.

All groups had 4 mice each. a. Detection of human DNA by FISH b. Detection of human c-Myc onco-protein by immunofluorescence. Statistical comparison between the xenograft bearing group and the two control groups, and that between the xenograft bearing group and the three anticancer treatment groups (CT, RT and Sx) was done by two-tailed student t-test. * < 0.05, ** < 0.01, *** <0.001, **** <0.0001. Statistical comparison between the three anti-cancer treatment groups (CT, RT and Sx) and those additionally treated with the three cfChPs deactivating agents was done by One-way ANOVA. * < 0.05, ** < 0.01.</p

Schema of the study design.

Metastatic dissemination following successful treatment of the primary tumour remains a common cause of death. There is mounting evidence that therapeutic interventions themselves may promote development of metastatic disease. We earlier reported that cell-free chromatin particles (cfChPs) released from dying cancer cells are potentially oncogenic. Based on this observation we hypothesized that therapeutic interventions may lead to the release of cfChPs from therapy induced dying cancer cells which could be carried via the blood stream to distant organs to transform healthy cells into new cancers that would masquerade as metastasis. To test this hypothesis, we generated xenografts of MDA-MB-231 human breast cancer cells in severe combined immune-deficient mice, and using immuno-fluorescence and FISH analysis looked for cfChPs in their brain cells. We detected multiple human DNA signals representing cfChPs in nuclei of brain cells of mice which co-localized with eight human onco-proteins. No intact MDA-MB-231 cells were detected. The number of co-localizing human DNA and human c-Myc signals increased dramatically following treatment with chemotherapy, localized radiotherapy or surgery, which could be prevented by concurrent treatment with three different cfChPs deactivating agents. These results suggest that therapeutic interventions lead to the release cfChPs from therapy induced dying cancer cells carrying oncogenes and are transported via the blood stream to brain cells to potentially transform them to generate new cancers that would appear as metastases. cfChPs induced metastatic spread of cancer is preventable by concurrent treatment with agents that deactivate cfChPs.</div

Figure showing fluorescently dual-labelled cfChPs in mouse brain (a and b).

Figure showing fluorescently dual-labelled cfChPs in mouse brain (a and b).</p