scholarly journals Cytokine Storm as a Cellular Response to dsDNA Breaks: A New Proposal

2021 ◽  
Vol 12 ◽  
Author(s):  
Snehal Shabrish ◽  
Indraneel Mittra
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
The Body ◽  
Host Cells ◽  
Preclinical Studies ◽  
Cytokine Storm ◽  
Vicious Cycle ◽  
Cascading Effect ◽  
Microbial Invasion ◽  
Cellular Dna

Pathogenesis of cytokine storm is poorly understood. In this article we propose a new mechanism and suggest innovative therapeutic avenues for its prevention. We have reported that particles of cell-free chromatin (cfCh) that are released from the billions of cells that die in the body everyday can illegitimately integrate into genomes of healthy cells to trigger dsDNA breaks. The latter leads to apoptosis and/or intense activation of inflammatory cytokines in the affected cells. We hypothesise that a similar phenomenon of dsDNA breaks and inflammation is involved in cytokine storm. The abundant cfCh particles that are released from dying host cells following viral/microbial invasion initiate a cascading effect of more cell death resulting in a vicious cycle of further DNA damage, apoptosis and hyper-inflammation which culminate in cytokine storm. We propose that this unrelenting vicious cycle of cellular DNA damage and cytokine storm may be the underlying cause of high mortality from severe COVID-19. We discuss results of our preclinical studies wherein we have shown that endotoxin induced cytokine storm in mice can be reversed by three different agents that have the ability to inactivate cfCh. These agents may be worthy of investigation in clinical trials to reduce mortality from COVID-19.

EXTH-06. ATR INHIBITORS AS MONOTHERAPY AND COMBINATORIAL THERAPY WITH TEMOZOLOMIDE IN PRECLINICAL GLIOBLASTOMA MODELS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi164-vi164
Author(s):  
Dimpy Koul ◽  
Xiaolong Li ◽  
Shaofang Wu ◽  
Sanjay Kumar singh ◽  
Kadir Caner Akdemir ◽  
...  
Keyword(s):  
Dna Damage ◽  
Combination Treatment ◽  
Cellular Response ◽  
Methylation Status ◽  
Standard Of Care ◽  
The Body ◽  
Treatment Modalities ◽  
Multiple Treatment ◽  
Sensitivity Pattern ◽  
Selective Sensitivity

Abstract Glioblastoma (GBM) is an aggressive brain tumor and has an extremely poor prognosis despite the use of multiple treatment modalities. DNA damage response (DDR) signaling plays an important role in inducing radiation and temozolomide (TMZ) resistance and hence has emerged as a molecular target for therapeutic development. The Ataxia Telangiectasia and Rad3-related protein (ATR) kinase is a key regulator of the DDR machinery, activated by DNA damage. Here, we show that three clinical-grade ATR inhibitors (Bay1895344, AZD6738 and Berzosertib) had similar selective sensitivity pattern across 16 glioma-like stem cell (GSC) lines tested. ATR inhibitors inhibited the growth of GSCs at low nanomolar range concentrations. Interestingly, all three ATR inhibitors showed a significant synergism with TMZ in a selective group of GSCs (Combination index and Bliss model). Importantly, we demonstrate that MGMT promoter methylation status was associated with cellular response to combination treatment with preferential inhibition of cell growth in MGMT promotor methylated (MGMT deficient) cell lines. Further, we compared the RNA-seq data from GSCs in the synergism and non-synergism group and used multiple complementary approaches to identify the response marker that confer sensitivity to combination therapy. Our preliminary data analysis identified several genes that confer sensitivity to combination treatment and studies are underway to validate the data. We also investigated the efficacy of BBB penetrant ATR inhibitor BAY 1895344 in orthotropic xenografts and administration of BAY 1895344 and TMZ combination significantly reduced tumor size and extended survival in an intracranial animal model. Combining ATR inhibitor with TMZ was well tolerated and did not confer additional toxicity as the body weights of TMZ and combination groups were comparable. The findings from this study provides a rationale to use ATR inhibitors in combination with TMZ in MGMT methylated tumors to improve therapeutic efficacy of standard of care for GBM patients.

scholarly journals A short HBV RNA region induces RNR-R2 expression in non-cycling cells and in primary human hepatocytes

2018 ◽  
Author(s):  
Inna Ricardo-Lax ◽  
Karin Broennimann ◽  
Julia Adler ◽  
Eleftherios Michailidis ◽  
Ype P de Jong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Dna Damage Response ◽  
Human Hepatocytes ◽  
Host Cells ◽  
Primary Human Hepatocytes ◽  
B Virus ◽  
Damage Response ◽  
Cellular Dna

AbstractHepatitis B virus infects non-dividing cells in which dNTPs are scarce. HBV replication requires dNTPs. To cope with this constraint the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels. Previously we reported that the HBx open reading frame (ORF) triggers this pathway. Unexpectedly however, we report here that the production of HBx protein is not essential. We found that a small region of 125 bases within the HBx transcript is sufficient to induce RNR-R2 expression in growth arrested HepG2 cells and in primary human hepatocytes (PHH). The observed HBx embedded regulatory element is named ERE. We demonstrate that ERE is functional as a positive strand RNA polymerase-II transcript. Remarkably, ERE is sufficient to induce the Chk1-E2F1-RNR-R2 DDR pathway, previously reported to be activated by HBV. Furthermore, we found that ERE activates ATR but not ATM in eliciting this DDR pathway in upregulating RNR-R2. These findings demonstrate the multitasking role of HBV transcripts in mediating virus-host cell interaction, a mechanism that explains how such a small genome effectively serves such a pervasive virus.Author summaryThe hepatitis B virus (HBV) infects the human liver and over 250 million people worldwide are chronically infected with HBV and at risk for cirrhosis and liver cancer. HBV has a very small DNA genome with only four genes, much fewer than other viruses. For propagation the virus consumes dNTPs, the building blocks of DNA, in much higher amounts than the infected cells provide. To cope with this constraint, the virus manipulates the cells to increase the production of dNTPs. We found that the virus activates the cellular response to DNA damage upon which the cells increase the production of dNTPs, but instead of repairing cellular DNA, the virus uses them for production of its own DNA. Usually viruses manipulate host cells with one or more of their unique proteins, however the small HBV genome cannot afford having such a unique gene and protein. Instead, we found that here the virus relies on RNA to manipulate the host cells. Our findings highlight the unprecedented principle of a multitasking viral RNA that is not only designed to be translated into proteins but also harbors an independent role in activating the cellular DNA damage response.

scholarly journals Involvement of Host ATR-CHK1 Pathway in Hepatitis B Virus Covalently Closed Circular DNA Formation

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jun Luo ◽  
Laurie Luckenbaugh ◽  
Hui Hu ◽  
Zhipeng Yan ◽  
Lu Gao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Replication ◽  
Host Cell ◽  
Hbv Infection ◽  
Host Cells ◽  
B Virus ◽  
Cellular Dna ◽  
Dna Processing

ABSTRACT The covalently closed circular (CCC) DNA of hepatitis B virus (HBV) functions as the only viral transcriptional template capable of producing all viral RNA species and is essential to initiate and sustain viral replication. CCC DNA is converted from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. As RC DNA mimics damaged cellular DNA, the host cell DNA damage repair (DDR) system is thought to be responsible for HBV CCC DNA formation. The potential role of two major cellular DDR pathways, the ataxia telangiectasia mutated (ATM) pathway and the ATM and Rad3-related (ATR) pathway, in HBV CCC DNA formation was thus investigated. Inhibition, or expression knockdown, of ATR and its downstream signaling factor CHK1, but not of ATM, decreased CCC DNA formation during de novo HBV infection, as well as intracellular CCC DNA amplification, when RC DNA from extracellular virions and intracellular nucleocapsids, respectively, is converted to CCC DNA. Furthermore, a novel RC DNA processing product with 5′ truncated minus strands was detected when the ATR-CHK1 pathway was inhibited, further indicating that this pathway controls RC DNA processing during its conversion to CCC DNA. These results provide new insights into how host cells recognize and process HBV RC DNA in order to produce CCC DNA and have implications for potential means to block CCC DNA production. IMPORTANCE Hepatitis B virus (HBV) chronically infects hundreds of millions of people and remains a major cause of viral hepatitis, cirrhosis, and liver cancer. HBV persistence is sustained by a viral nuclear episome that directs all viral gene expression needed to support viral replication. The episome is converted from an incomplete DNA precursor in viral particles in an ill-understood process. We report here that the incomplete DNA precursor is recognized by the host cell in a way similar to the sensing of damaged cellular DNA for subsequent repair to form the nuclear episome. Intense efforts are ongoing to develop novel antiviral strategies to eliminate CCC DNA so as to cure chronic HBV infection. Our results here provide novel insights into, and suggest novel ways of perturbing, the process of episome formation. Furthermore, our results inform mechanisms of cellular DNA damage recognition and repair, processes essential for normal cell growth.

scholarly journals Viral Transport of DNA Damage That Mimics a Stalled Replication Fork

2005 ◽  
Vol 79 (1) ◽  
pp. 569-580 ◽  
Author(s):  
Jaana Jurvansuu ◽  
Kenneth Raj ◽  
Andrzej Stasiak ◽  
Peter Beard
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Cellular Response ◽  
Replication Fork ◽  
Dna Polymerase Delta ◽  
Viral Dna ◽  
Focus Formation ◽  
Viral Dna Replication ◽  
Cross Links ◽  
Cellular Dna

ABSTRACT Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.

scholarly journals Cytolethal Distending Toxin: from mitotic DNA damage to cGAS-dependent pro-inflammatory response

2020 ◽  
Author(s):  
Benoît J. Pons ◽  
Aurélie Pettes-Duler ◽  
Claire Naylies ◽  
Frédéric Taieb ◽  
Saleha Hashim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cellular Response ◽  
Cell Cycle Checkpoints ◽  
Host Cells ◽  
Cytolethal Distending Toxin ◽  
Dna Damaging Agents ◽  
Damage Response ◽  
Precise Relationship ◽  
First Time

AbstractThe Cytolethal Distending Toxin (CDT) is a bacterial genotoxin that activates the DNA damage response and induces inflammatory signatures in host cells, but the precise relationship between these outcomes has not been addressed so far. CDT induces a singular time-dependent increase of DNA damage and cell cycle defects, questioning on possible impaired response to this toxin over the cell cycle. Here, we identify mitosis as a crucial phase during CDT intoxination. Despite active cell cycle checkpoints and in contrast to other DNA damaging agents, CDT-exposed cells reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation. These micronuclei are recognized by cGAS that elicits an inflammatory signature resulting in cell distention and senescence. Our results unravel for the first time the mitotic consequences of CDT genotoxic activity and relate them to pro-inflammatory cellular response. These findings may have important implications during bacterial infection regarding CDT-mediated immunomodulatory and tumorigenic processes.

scholarly journals A phosphorylation-dependent switch in the disordered p53 transactivation domain regulates DNA binding

2020 ◽  
Vol 118 (1) ◽  
pp. e2021456118
Author(s):  
Xun Sun ◽  
H. Jane Dyson ◽  
Peter E. Wright
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Posttranslational Modifications ◽  
Cellular Response ◽  
Transactivation Domain ◽  
Tumor Suppressor P53 ◽  
Rich Domain ◽  
Binding Surface ◽  
Cellular Dna ◽  
Binding Curve

The tumor-suppressor p53 is a critical regulator of the cellular response to DNA damage and is tightly regulated by posttranslational modifications. Thr55 in the AD2 interaction motif of the N-terminal transactivation domain functions as a phosphorylation-dependent regulatory switch that modulates p53 activity. Thr55 is constitutively phosphorylated, becomes dephosphorylated upon DNA damage, and is subsequently rephosphorylated to facilitate dissociation of p53 from promoters and inactivate p53-mediated transcription. Using NMR and fluorescence spectroscopy, we show that Thr55 phosphorylation inhibits DNA-binding by enhancing competitive interactions between the disordered AD2 motif and the structured DNA-binding domain (DBD). Nonphosphorylated p53 exhibits positive cooperativity in binding DNA as a tetramer. Upon phosphorylation of Thr55, cooperativity is abolished and p53 binds initially to cognate DNA sites as a dimer. As the concentration of phosphorylated p53 is further increased, a second dimer binds and causes p53 to dissociate from the DNA, resulting in a bell-shaped binding curve. This autoinhibition is driven by favorable interactions between the DNA-binding surface of the DBD and the multiple phosphorylated AD2 motifs within the tetramer. These interactions are augmented by additional phosphorylation of Ser46 and are fine-tuned by the proline-rich domain (PRD). Removal of the PRD strengthens the AD2–DBD interaction and leads to autoinhibition of DNA binding even in the absence of Thr55 phosphorylation. This study reveals the molecular mechanism by which the phosphorylation status of Thr55 modulates DNA binding and controls both activation and termination of p53-mediated transcriptional programs at different stages of the cellular DNA damage response.

scholarly journals Campylobacter jejuni Cytolethal Distending Toxin Promotes DNA Repair Responses in Normal Human Cells

2003 ◽  
Vol 71 (1) ◽  
pp. 541-545 ◽  
Author(s):  
Duane C. Hassane ◽  
Robert B. Lee ◽  
Carol L. Pickett
Keyword(s):  
Dna Damage ◽  
Campylobacter Jejuni ◽  
Cellular Response ◽  
Human Fibroblasts ◽  
Host Cells ◽  
Cytolethal Distending Toxin ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Exogenous Addition ◽  
Normal Human

ABSTRACT Cytolethal distending toxin (CDT) is a multisubunit protein found in various gram-negative bacterial pathogens of humans which is thought to cause cell death by direct DNA damage of host cells. We sought to determine if a cellular response to DNA damage could be detected by exogenous addition of the holotoxin. Exogenous addition of the Campylobacter jejuni 81-176 CDT to primary human fibroblasts resulted in formation of Rad50 foci, which are formed around double-stranded-DNA breaks. Moreover, such foci are formed in both proliferating and nonproliferating cells that are treated with C. jejuni CDT. Fibroblasts that were intoxicated and later stimulated to proliferate failed to divide and remained arrested in the G1 phase of the cell cycle.

scholarly journals DNA-damage response pathways triggered by viral replication

2006 ◽  
Vol 8 (5) ◽  
pp. 1-11 ◽  
Author(s):  
Alison Sinclair ◽  
Sarah Yarranton ◽  
Celine Schelcher
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Viral Replication ◽  
Dna Damage Response ◽  
Cellular Response ◽  
Viral Infections ◽  
Herpes Simplex Virus 1 ◽  
Barr Virus ◽  
Damage Response ◽  
Cellular Dna

Many viruses, with distinct replication strategies, activate DNA-damage response pathways, including the lentivirus human immunodeficiency virus (HIV) and the DNA viruses Epstein–Barr virus (EBV), herpes simplex virus 1, adenovirus and SV40. DNA-damage response pathways involving DNA-dependent protein kinase, ataxia-telengiectasia mutated (ATM) and ‘ataxia-telengiectasia and Rad3-related’ (ATR) have all been implicated. This review focuses on the effects of HIV and EBV replication on DNA repair pathways. It has been suggested that activation of cellular DNA repair and recombination enzymes is beneficial for viral replication, as illustrated by the ability of suppressors of the ATM and ATR family to inhibit HIV replication. However, activation of DNA-damage response pathways can also promote apoptosis. Viruses can tailor the cellular response by suppressing downstream signalling from DNA-damage sensors, as exemplified by EBV. New small-molecule inhibitors of the DNA-damage response pathways could therefore be of value to treat viral infections.

scholarly journals DNA repair in ischemic acute kidney injury

2017 ◽  
Vol 312 (4) ◽  
pp. F551-F555 ◽  
Author(s):  
Jeffrey D. Pressly ◽  
Frank Park
Keyword(s):  
Dna Damage ◽  
Acute Kidney Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Kidney Injury ◽  
The Body ◽  
Negative Effects ◽  
Dna Damage And Repair ◽  
Dna Strand ◽  
Cellular Dna

Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury leading to an induction of oxidative stress, cellular dysfunction, and loss of renal function. DNA damage, including oxidative base modifications and physical DNA strand breaks, is a consequence of renal IRI. Like many other organs in the body, a redundant and highly conserved set of endogenous repair pathways have evolved to selectively recognize the various types of cellular DNA damage and combat its negative effects on cell viability. Severe damage to the DNA, however, can trigger cell death and elimination of the injured tubular epithelial cells. In this minireview, we summarize the state of the current field of DNA damage and repair in the kidney and provide some expected and, in some cases, unexpected effects of IRI on DNA damage and repair in the kidney. These findings may be applicable to other forms of acute kidney injury and could provide new opportunities for renal research.

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