Biology subjects

Khare, N. K.

Publications and source records attributed to Khare, N. K..

8 recordsLinked to original sources

bioRxiv2026

Circulating Cell-Free Chromatin Particles Trigger a Unique Biphasic STING Signaling Program that Drives DNA Damage and Inflammation

Cell death, DNA damage, and inflammation are closely interconnected processes implicated in ageing, cancer, and inflammatory disorders, yet the endogenous mechanisms linking them remain unclear. We previously identified cell-free chromatin particles (cfChPs), released from dying cells, as biologically active entities that enter neighboring cells and induce DNA damage and inflammation. Here, we show that serum-derived circulating cfChPs are rapidly internalized by human peripheral blood mononuclear cells and trigger a previously unrecognized biphasic STING signaling response. An early phase involves rapid STING trafficking to the perinuclear region and nucleus, with activation of IRF3 and NF-{kappa}B preceding detectable DNA damage. This is followed by a later phase characterized by STING phosphorylation, puncta formation, persistent DNA damage, and robust inflammatory cytokine production. Pharmacological inhibition or genetic deletion of STING markedly attenuated these responses. These findings identify extracellular cfChPs as endogenous DNA-damaging agents and reveal biphasic STING signaling as a mechanistic link between cell death, DNA damage, and sterile inflammation. One-sentence summaryCirculating cell-free chromatin particles released from dying cells trigger a previously unrecognized biphasic STING signaling program that mechanistically links cell death to DNA damage and sterile inflammation, providing a potential basis for aging, cancer, and inflammatory diseases.

immunology↗

bioRxiv2024

Induction of pluripotent oncogenic stem cells from mouse fibroblasts

Natural biological agents that can transform normal somatic cells into cancer stem cells, have not been identified. We earlier reported that cell free chromatin particles (cfChPs) that circulate in blood of cancer patients can horizontally transfer themselves to healthy cells to induce dsDNA breaks and inflammation. Here we show that a single cell clone D5 developed from NIH3T3 mouse fibroblast cells treated with cfChPs isolated from sera of cancer patients exhibited upregulation of stemness related transcription factors and surface markers, and the ability to form spheroids in appropriate culture medium. Transcriptome analysis revealed upregulation of cancer related pathways including invasion, metastasis and stemness. Subcutaneous inoculation into SCID mice resulted in development of malignant tumors which expressed all three germline markers. Our results suggest the cfChPs that circulate in blood of cancer patients are oncogenic and can transform susceptible somatic cells into cancer stem cells with the potential to promote metastasis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/622204v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@4288org.highwire.dtl.DTLVardef@1102d4borg.highwire.dtl.DTLVardef@10daaf6org.highwire.dtl.DTLVardef@53084d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Cell-free chromatin particles damage genomic DNA of healthy cells via an ROS-independent mechanism

Several hundred billion cells die daily in the human body, releasing cell-free chromatin particles (cfChPs) in the process, which then enter the blood circulation and get taken up by healthy cells. We have previously reported that, these internalized cfChPs damage genomic DNA as well cause physical damage to mitochondria, resulting in increased mitochondrial ROS production. In the current study, we evaluated the potential damaging effects of the cfChP-induced increase in ROS production on genomic DNA. NIH3T3 mouse fibroblast cells were treated with cfChPs isolated from the sera of healthy individuals (H-cfChPs) or patients with cancer (C-cfChPs) in the presence or absence of the ROS scavenger Mito-TEMPO. The pre-incubation of cfChP-treated cells with Mito-TEMPO abolished ROS production, but did not prevent genomic DNA damage induced by H-cfChPs and C-cfChPs. Our results suggest that cfChP-induced genomic DNA damage occurs in an ROS-independent manner. These findings align with emerging evidence suggesting that mitochondrial ROS may not be a direct cause of genomic DNA damage and suggest that DNA damage attributed to ROS may in-fact be induced by cfChPs. This possibility opens up new therapeutic approaches involving deactivation of cfChPs to retard ageing and other degenerative conditions traditionally attributed to oxidative stress.

cell biology↗

bioRxiv2023

Horizontally transferred cell-free chromatin particles function as miniature predatory genomes and vehicles for transposable elements within host cells

BackgroundHorizontal gene transfer (HGT) plays an important evolutionary role in prokaryotes, but it is thought to be less frequent in mammals. We previously reported that cell-free chromatin particles (cfChPs) - chromosomal fragments released from the billions of dying cells that circulate in human blood - are horizontally transferred to healthy cells with biological effects. However, the underlying mechanism and function of these effects remained unclear. MethodsWe treated NIH3T3 mouse fibroblasts cells with cfChPs isolated from human serum and serially passaged the cells. The intracellular activities of cfChPs were analysed using chromatin fibre fluorography, cytogenetic analysis, immuno-fluorescence and fluorescent in situ hybridisation. ResultsWe discovered that the internalised cfChPs were almost exclusively comprised of non-coding DNA, and the disparate DNA sequences contained within them had randomly combined to form complex concatemers some of which were ostensibly multi-mega base pairs in size. The concatemers exhibited variable and bizarre spatial relationships with the host cell interphase DNA with many remaining in the cytoplasm and others aligning themselves with the mouse chromosomal DNA. The concatemers autonomously performed many functions attributable to the nuclear genome. Being associated with DNA polymerase, the concatemers could synthesize DNA and autonomously replicate themselves without heed to the mitotic cycle of the host cell. They could synthesise RNA, RNA polymerase, ribosomal RNA, ribosomal proteins, and generate numerous human proteins, including oncogenes, within the mouse cells which manifested as complex and highly amplified multi-peptide fusion proteins. The concatemers harboured human LINE-1 and Alu elements, which being associated with DNA polymerase, reverse transcriptase and transposes could markedly amplify themselves and increase their copy number with time in culture with the potential to rearrange themselves within the mouse genome. The above findings were reproducible in four other cell lines derived from different species suggesting that horizontal transfer of cfChPs may be a universal phenomenon. ConclusionsOur results lead us to propose that: 1) a cell simultaneously harbours two autonomous genome forms: one that is inherited (hereditary genome) and numerous others that are acquired (satellite genomes); 2) satellite genomes may potentially have evolutionary functions given their ability to serve as vehicles for transposable elements and to generate a plethora of novel proteins; 3) transposable elements are "foreign" genetic elements that are acquired from dying cells via HGT; 4) non-coding DNA has many hidden biological functions that remain dormant but are activated following cellular apoptosis to become detectable in association with the cfChP concatemers; 5) "within-self" HGT occurs in mammals on a massive scale via the medium of cfChP concatemers that have undergone extensive and complex modifications resulting in their behaviour as "foreign" genetic elements. Video Abstracthttps://drive.google.com/file/d/1I6NbrYmT9BRM63ywYj47AU0O9WJ_NrcR/view?usp=drive_link

evolutionary biology↗

bioRxiv2023

Metastases arise as new cancers from cells of target organs transformed by cell-free chromatin particles released from dying cancer cells

IntroductionBased on our earlier finding that cell-free chromatin particles (cfChPs) released from dying cancer cells are potentially oncogenic, we hypothesised that metastases arise as new cancers from the cells of target organs transformed by cfChPs released from dying cancer cells. MethodsWe fluorescently dually-labelled MDA-MB-231 human breast cancer cells and A-375 human melanoma cells in their DNA with BrdU and in their histones with CellLight(R) Histone 2BGFP. One hundred thousand dually-labelled cells were intravenously injected into SCID mice. In other experiments unlabelled cells were injected for detection of lung metastasis. Also intravenously injected were 700 ng of purified cfChPs isolated from radiation-killed MDA-MB-231 cells. ResultsWe observed that the fluorescently dually-labelled MDA-MB-231 and A-375 cells died upon reaching the lungs and released dually-labelled fluorescent chromatin particles that accumulated in the nuclei of lung cells at 48 h. Injection of unlabelled cfChPs led to the activation of 10 hallmarks of cancer and immune checkpoints in lung cells at 72 h, suggesting that the lung cells had rapidly transformed into incipient cancer cells. Fluorescent in situ hybridisation analysis of lung metastases that subsequently developed using mouse and human specific DNA probes revealed that the tumour cells contained both mouse and human DNA in almost equal proportions. Similarly, immune-fluorescence analysis using species specific mAbs revealed that the tumour cells co-expressed mouse and human specific proteins. Metaphase preparations and single-cell clones developed from cell cultures of lung metastases were found to contain chimeric chromosomes containing both mouse and human DNA, and the cells to co-express both human- and mouse-specific proteins. The Intravenously injected purified cfChPs isolated from radiation-killed MDA-MB-231 cells also induced lung metastasis which predominantly contained mouse DNA strongly suggesting that the metastatic tumours had arisen from the mouse lung cells. ConclusionThese results provide strong evidence that cfChPs released from dying cancer cells integrate into genomes of target lung cells to transform them into new cancers that masquerade as metastasis. They support our hypothesis that metastases arise from the cells of target organs and not from those of the primary tumour. These findings have implications for the principles of cancer therapy.

cancer biology↗

bioRxiv2023

Cell-free chromatin from dying cells trigger an immune response via the cGAS-STING pathway

It is long established that cell death and immune response are closely related, although the nature of this relationship has remained unclear. We earlier reported that cell-free chromatin particles (cfChPs) released from the billions of cells that die in the body every day to enter into the blood circulation, or those that are released locally from dying cells, can readily enter into healthy cells to induce DNA damage and activate inflammatory cytokines. In this study we investigated whether cfChPs from dying cells might be the missing link between cell death and immune response. We treated human lymphocytes with cfChPs isolated from sera of healthy individuals or with cfChPs released from hypoxia-induced dying lymphocytes. We observed that cfChPs from both sources readily entered into lymphocytes to accumulate in their nuclei within 2 h. This was associated with marked activation of CD69 and release of inflammatory cytokines, as well as of p-STING and cGAS expression. The addition of the STING protein inhibitor H151 to the cfChPs treated cells abolished the release of inflammatory cytokines. These findings lead us to suggest that cfChPs from dying cells are the critical triggers of immune response which act via cGAS-STING pathway.

immunology↗

bioRxiv2023

Cell-free chromatin particles activate immune checkpoints in human T cells: Implications for cancer therapy

Immune checkpoint blockade is an exciting breakthrough in cancer therapy, but how immune checkpoints are activated is unknown. We have earlier reported that cell-free chromatin particles (cfChPs) that circulate in the blood, or those that are released locally from dying cells, are readily internalized by healthy cells with biological consequences. Here we show that treatment of human lymphocytes with cfChPs isolated from sera of cancer patients led to marked activation of immune checkpoints viz. PD-1, CTLA-4, LAG-3, NKG2A, and TIM-3. Concurrently activated were stress-related markers cJun, cFos, JunB, FosB, NF[Kcy]B, and EGR1. The above immune checkpoints were also activated when lymphocytes were treated with cfChPs released from dying HeLa cells; the latter could be abrogated by three cfChPs deactivating agents. These results suggest that immune checkpoints are activated by lymphocytes as stress response to cfChPs. Simultaneous downregulation of multiple immune checkpoints may herald a new approach to immunotherapy of cancer. Statement of SignificanceWe show that cell-free chromatin particles (cfChPs) that circulate in the blood of cancer patients, or those released from dying cancer cells, simultaneously activate five immune checkpoints as a stress response by human lymphocytes. Activation of checkpoints was abrogated by cfChPs deactivating agents suggesting a novel approach to cancer treatment.

immunology↗

bioRxiv2021

Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells

mtDNA damage and the resultant oxidative stress are associated with neurodegenerative diseases, ageing and cancer. However, what triggers mtDNA damage remains unclear. We have reported that cell-free chromatin particles (cfChPs) that are released from the billions of cells that die in the body every day can readily enter into healthy cells and damage their DNA. We show here that cfChPs isolated from sera of healthy individuals, or those that are released from dying cells, inflict direct physical damage mtDNA leading to marked activation of ROS. The latter could be abrogated by concurrent treatment with three different cfChPs deactivating agents. Given that 1x109-1x1012 cells die in the body every day, our findings suggest that cfChPs from dying cells are major physiological triggers for mtDNA damage and ROS production. Deactivation of cfChPs may provide a novel therapeutic approach to retard ageing and associated degenerative conditions that have been linked to oxidative stress.

biochemistry↗