NBS1-CtIP-mediated DNA end resection suppresses cGAS binding to micronuclei.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is activated in cells with defective DNA damage repair and signaling (DDR) factors, but a direct role for DDR factors in regulating cGAS activation in response to micronuclear DNA is still poorly understood. Here, we provide novel evidence that Nijmegen breakage syndrome 1 (NBS1) protein, a well-studied DNA double-strand break (DSB) sensor-in coordination with Ataxia Telangiectasia Mutated (ATM), a protein kinase, and Carboxy-terminal binding protein 1 interacting protein (CtIP), a DNA end resection factor-functions as an upstream regulator that prevents cGAS from binding micronuclear DNA. When NBS1 binds to micronuclear DNA via its fork-head-associated domain, it recruits CtIP and ATM via its N- and C-terminal domains, respectively. Subsequently, ATM stabilizes NBS1's interaction with micronuclear DNA, and CtIP converts DSB ends into single-strand DNA ends; these two key events prevent cGAS from binding micronuclear DNA. Additionally, by using a cGAS tripartite system, we show that cells lacking NBS1 not only recruit cGAS to a major fraction of micronuclear DNA but also activate cGAS in response to these micronuclear DNA. Collectively, our results underscore how NBS1 and its binding partners prevent cGAS from binding micronuclear DNA, in addition to their classical functions in DDR signaling.

Carbon Ion Radiotherapy in the Management of Hepatocellular Carcinoma.

Localized hepatocellular carcinoma (HCC) that is unresectable and non-transplantable can be treated by several liver-directed therapies. External beam radiation therapy (EBRT) is an increasingly accepted and widely utilized treatment modality in this setting. Accelerated charged particles such as proton beam therapy (PBT) and carbon ion radiation therapy (CIRT) offer technological advancements over conventional photon radiotherapy. In this review, we summarize the distinct advantages of CIRT use for HCC treatment, focusing on physical and biological attributes, and outline dosimetric and treatment planning caveats. Based on these considerations, we posit that HCC may be among the best indications for use of CIRT, as it allows for maximizing tumoricidal doses to the target volume while minimizing the dose to the organs at risk.

Immunomodulatory Effects of Radiotherapy.

Radiation therapy (RT), an integral component of curative treatment for many malignancies, can be administered via an increasing array of techniques. In this review, we summarize the properties and application of different types of RT, specifically, conventional therapy with x-rays, stereotactic body RT, and proton and carbon particle therapies. We highlight how low-linear energy transfer (LET) radiation induces simple DNA lesions that are efficiently repaired by cells, whereas high-LET radiation causes complex DNA lesions that are difficult to repair and that ultimately enhance cancer cell killing. Additionally, we discuss the immunogenicity of radiation-induced tumor death, elucidate the molecular mechanisms by which radiation mounts innate and adaptive immune responses and explore strategies by which we can increase the efficacy of these mechanisms. Understanding the mechanisms by which RT modulates immune signaling and the key players involved in modulating the RT-mediated immune response will help to improve therapeutic efficacy and to identify novel immunomodulatory drugs that will benefit cancer patients undergoing targeted RT.

Dysfunctional telomeres trigger cellular senescence mediated by cyclic GMP-AMP synthase.

Defective DNA damage response (DDR) signaling is a common mechanism that initiates and maintains the cellular senescence phenotype. Dysfunctional telomeres activate DDR signaling, genomic instability, and cellular senescence, but the links among these events remains unclear. Here, using an array of biochemical and imaging techniques, including a highly regulatable CRISPR/Cas9 strategy to induce DNA double strand breaks specifically in the telomeres, ChIP, telomere immunofluorescence, fluorescence in situ hybridization (FISH), micronuclei imaging, and the telomere shortest length assay (TeSLA), we show that chromosome mis-segregation due to imperfect DDR signaling in response to dysfunctional telomeres creates a preponderance of chromatin fragments in the cytosol, which leads to a premature senescence phenotype. We found that this phenomenon is caused not by telomere shortening, but by cyclic GMP-AMP synthase (cGAS) recognizing cytosolic chromatin fragments and then activating the stimulator of interferon genes (STING) cytosolic DNA-sensing pathway and downstream interferon signaling. Significantly, genetic and pharmacological manipulation of cGAS not only attenuated immune signaling, but also prevented premature cellular senescence in response to dysfunctional telomeres. The findings of our study uncover a cellular intrinsic mechanism involving the cGAS-mediated cytosolic self-DNA-sensing pathway that initiates premature senescence independently of telomere shortening.

Isolation of time-dependent DNA damage induced by energetic carbon ions and their fragments using fluorescent nuclear track detectors.

PURPOSE: High energetic carbon (C-) ion beams undergo nuclear interactions with tissue, producing secondary nuclear fragments. Thus, at depth, C-ion beams are composed of a mixture of different particles with different linear energy transfer (LET) values. We developed a technique to enable isolation of DNA damage response (DDR) in mixed radiation fields using beam line microscopy coupled with fluorescence nuclear track detectors (FNTDs). METHODS: We imaged live cells on a coverslip made of FNTDs right after C-ion, proton or photon irradiation using an in-house built confocal microscope placed in the beam path. We used the FNTD to link track traversals with DNA damage and separated DNA damage induced by primary particles from fragments. RESULTS: We were able to spatially link physical parameters of radiation tracks to DDR in live cells to investigate spatiotemporal DDR in multi-ion radiation fields in real time, which was previously not possible. We demonstrated that the response of lesions produced by the high-LET primary particles associates most strongly with cell death in a multi-LET radiation field, and that this association is not seen when analyzing radiation induced foci in aggregate without primary/fragment classification. CONCLUSIONS: We report a new method that uses confocal microscopy in combination with FNTDs to provide submicrometer spatial-resolution measurements of radiation tracks in live cells. Our method facilitates expansion of the radiation-induced DDR research because it can be used in any particle beam line including particle therapy beam lines. CATEGORY: Biological Physics and Response Prediction.

Nonhomologous End Joining Is More Important Than Proton Linear Energy Transfer in Dictating Cell Death.

PURPOSE: This study seeks to identify biological factors that may yield a therapeutic advantage of proton therapy versus photon therapy. Specifically, we address the role of nonhomologous end-joining (NHEJ) and homologous recombination (HR) in the survival of cells in response to clinical photon and proton beams. METHODS AND MATERIALS: We irradiated HT1080, M059K (DNA-PKcs+/+), and HCC1937 human cancer cell lines and their isogenic counterparts HT1080-shDNA-PKcs, HT1080-shRAD51IND, M059J (DNA-PKcs-/-), and HCC1937-BRCA1 (BRCA1 complemented) to assess cell clonogenic survival and γ-H2AX radiation-induced foci. Cells were irradiated with either clinically relevant photons or 1 of 3 proton linear energy transfer (LET) values. RESULTS: Our results indicate that NHEJ deficiency is more important in dictating cell survival than proton LET. Cells with disrupted HR through BRCA1 mutation showed increased radiosensitivity only for high-LET protons whereas RAD51 depletion showed increased radiosensitivity for both photons and protons. DNA double strand breaks, assessed by γ-H2AX radiation-induced foci, showed greater numbers after 24 hours in cells exposed to higher LET protons. We also observed that NHEJ-deficient cells were unable to repair the vast majority of double strand breaks after 24 hours. CONCLUSIONS: BRCA1 mutation significantly sensitizes cells to protons, but not photons. Loss of NHEJ renders cells hypersensitive to radiation, whereas the relative importance of HR increases with LET across several cell lines. This may be attributable to the more clustered damage induced by higher LET protons, which are harder to repair through NHEJ. This highlights the importance of tumor biology in dictating treatment modality and suggests BRCA1 as a potential biomarker for proton therapy response. Our data also support the use of pharmacologic inhibitors of DNA repair to enhance the sensitivity to different radiation types, although this raises issues for normal tissue toxicity.

Nuclear Foci Assays in Live Cells.

DNA double strand breaks (DSBs) are a serious threat to genome stability and cell viability. Accurate detection of DSBs is critical for the basic understanding of cellular response to ionizing radiation. Recruitment and retention of DNA repair and response proteins at DSBs can be conveniently visualized by fluorescence imaging (often called ionizing radiation-induced foci) both in live and fixed cells. In this chapter, we describe a live cell imaging methodology that directly monitors induction and repair of single DSB, recruitment kinetics of DSB repair/sensor factors to DSB sites, and dynamic interaction of DSB repair/sensor proteins with DSBs at single-cell level. Additionally, the methodology described in this chapter can be readily adapted to other DSBs repair/sensor factors and cell types.

Mechanistic link between DNA damage sensing, repairing and signaling factors and immune signaling.

Previously, DNA damage sensing, repairing and signaling machineries were thought to mainly suppress genomic instability in response to genotoxic stress. Emerging evidence indicates a crosstalk between DNA repair machinery and the immune system. In this chapter, we attempt to decipher the molecular choreography of how factors, including ATM, BRCA1, DNA-PK, FANCA/D2, MRE11, MUS81, NBS1, RAD51 and TREX1, of multiple DNA metabolic processes are directly or indirectly involved in suppressing cytosolic DNA sensing pathway-mediated immune signaling. We provide systematic details showing how different DDR factors' roles in modulating immune signaling are not direct, but are rather a consequence of their inherent ability to sense, repair and signal in response to DNA damage. Unexpectedly, most DDR factors negatively impact the immune system; that is, the immune system shows defective signaling if there are defects in DNA repair pathways. Thus, in addition to their known DNA repair and replication functions, DDR factors help prevent erroneous activation of immune signaling. A more precise understanding of the mechanisms by which different DDR factors function in immune signaling can be exploited to redirect the immune system for both preventing and treating autoimmunity, cellular senescence and cancer in humans.

Werner Syndrome Protein and DNA Replication.

Werner Syndrome (WS) is an autosomal recessive disorder characterized by the premature development of aging features. Individuals with WS also have a greater predisposition to rare cancers that are mesenchymal in origin. Werner Syndrome Protein (WRN), the protein mutated in WS, is unique among RecQ family proteins in that it possesses exonuclease and 3' to 5' helicase activities. WRN forms dynamic sub-complexes with different factors involved in DNA replication, recombination and repair. WRN binding partners either facilitate its DNA metabolic activities or utilize it to execute their specific functions. Furthermore, WRN is phosphorylated by multiple kinases, including Ataxia telangiectasia mutated, Ataxia telangiectasia and Rad3 related, c-Abl, Cyclin-dependent kinase 1 and DNA-dependent protein kinase catalytic subunit, in response to genotoxic stress. These post-translational modifications are critical for WRN to function properly in DNA repair, replication and recombination. Accumulating evidence suggests that WRN plays a crucial role in one or more genome stability maintenance pathways, through which it suppresses cancer and premature aging. Among its many functions, WRN helps in replication fork progression, facilitates the repair of stalled replication forks and DNA double-strand breaks associated with replication forks, and blocks nuclease-mediated excessive processing of replication forks. In this review, we specifically focus on human WRN's contribution to replication fork processing for maintaining genome stability and suppressing premature aging. Understanding WRN's molecular role in timely and faithful DNA replication will further advance our understanding of the pathophysiology of WS.

RAD51 interconnects between DNA replication, DNA repair and immunity.

RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity.

Repurposing DNA repair factors to eradicate tumor cells upon radiotherapy.

Cancer is the leading cause of death worldwide. Almost 50% of all cancer patients undergo radiation therapy (RT) during treatment, with varying success. The main goal of RT is to kill tumor cells by damaging their DNA irreversibly while sparing the surrounding normal tissue. The outcome of RT is often determined by how tumors recognize and repair their damaged DNA. A growing body of evidence suggests that tumors often show abnormal expression of DNA double-strand break (DSB) repair genes that are absent from normal cells. Defects in a specific DNA repair pathway make tumor cells overly dependent on alternative or backup pathways to repair their damaged DNA. These tumor cell-specific abnormalities in the DNA damage response (DDR) machinery can potentially be used as biomarkers for treatment outcomes or as targets for sensitization to ionizing radiation (IR). An improved understanding of genetic or epigenetic alterations in the DNA repair pathways specific to cancer cells has paved the way for new treatments that combine pharmacological exploitation of tumor-specific molecular vulnerabilities with IR. Inhibiting DNA repair pathways has the potential to greatly enhance the therapeutic ratio of RT. In this review, we will discuss DNA repair pathways in active cells and how these pathways are deregulated in tumors. We will also describe the impact of targeting cancer-specific aberrations in the DDR as a treatment strategy to improve the efficacy of RT. Finally, we will address the current roadblocks and future prospects of these approaches.

Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments.

Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability.

Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells.

PURPOSE: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury. METHODS AND MATERIALS: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10(6) protons/cm(2). RESULTS: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. CONCLUSIONS: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.

Evaluating biomarkers to model cancer risk post cosmic ray exposure.

Robust predictive models are essential to manage the risk of radiation-induced carcinogenesis. Chronic exposure to cosmic rays in the context of the complex deep space environment may place astronauts at high cancer risk. To estimate this risk, it is critical to understand how radiation-induced cellular stress impacts cell fate decisions and how this in turn alters the risk of carcinogenesis. Exposure to the heavy ion component of cosmic rays triggers a multitude of cellular changes, depending on the rate of exposure, the type of damage incurred and individual susceptibility. Heterogeneity in dose, dose rate, radiation quality, energy and particle flux contribute to the complexity of risk assessment. To unravel the impact of each of these factors, it is critical to identify sensitive biomarkers that can serve as inputs for robust modeling of individual risk of cancer or other long-term health consequences of exposure. Limitations in sensitivity of biomarkers to dose and dose rate, and the complexity of longitudinal monitoring, are some of the factors that increase uncertainties in the output from risk prediction models. Here, we critically evaluate candidate early and late biomarkers of radiation exposure and discuss their usefulness in predicting cell fate decisions. Some of the biomarkers we have reviewed include complex clustered DNA damage, persistent DNA repair foci, reactive oxygen species, chromosome aberrations and inflammation. Other biomarkers discussed, often assayed for at longer points post exposure, include mutations, chromosome aberrations, reactive oxygen species and telomere length changes. We discuss the relationship of biomarkers to different potential cell fates, including proliferation, apoptosis, senescence, and loss of stemness, which can propagate genomic instability and alter tissue composition and the underlying mRNA signatures that contribute to cell fate decisions. Our goal is to highlight factors that are important in choosing biomarkers and to evaluate the potential for biomarkers to inform models of post exposure cancer risk. Because cellular stress response pathways to space radiation and environmental carcinogens share common nodes, biomarker-driven risk models may be broadly applicable for estimating risks for other carcinogens.

Ionizing radiation and heart risks.

Cardiovascular disease and cancer are the two leading causes of morbidity and mortality worldwide. As advancements in radiation therapy (RT) have significantly increased the number of cancer survivors, the risk of radiation-induced cardiovascular disease (RICD) in this group is a growing concern. Recent epidemiological data suggest that accidental or occupational exposure to low dose radiation, in addition to therapeutic ionizing radiation, can result in cardiovascular complications. The progression of radiation-induced cardiotoxicity often takes years to manifest but is also multifaceted, as the heart may be affected by a variety of pathologies. The risk of cardiovascular disease development in RT cancer survivors has been known for 40 years and several risk factors have been identified in the last two decades. However, most of the early work focused on clinical symptoms and manifestations, rather than understanding cellular processes regulating homeostatic processes of the cardiovascular system in response to radiation. Recent studies have suggested that a different approach may be needed to refute the risk of cardiovascular disease following radiation exposure. In this review, we will focus on how different radiation types and doses may induce cardiovascular complications, highlighting clinical manifestations and the mechanisms involved in the pathophysiology of radiation-induced cardiotoxicity. We will finally discuss how current and future research on heart development and homeostasis can help reduce the incidence of RICD.

Replication stress induced site-specific phosphorylation targets WRN to the ubiquitin-proteasome pathway.

Faithful and complete genome replication in human cells is essential for preventing the accumulation of cancer-promoting mutations. WRN, the protein defective in Werner syndrome, plays critical roles in preventing replication stress, chromosome instability, and tumorigenesis. Herein, we report that ATR-mediated WRN phosphorylation is needed for DNA replication and repair upon replication stress. A serine residue, S1141, in WRN is phosphorylated in vivo by the ATR kinase in response to replication stress. ATR-mediated WRN S1141 phosphorylation leads to ubiquitination of WRN, facilitating the reversible interaction of WRN with perturbed replication forks and subsequent degradation of WRN. The dynamic interaction between WRN and DNA is required for the suppression of new origin firing and Rad51-dependent double-stranded DNA break repair. Significantly, ATR-mediated WRN phosphorylation is critical for the suppression of chromosome breakage during replication stress. These findings reveal a unique role for WRN as a modulator of DNA repair, replication, and recombination, and link ATR-WRN signaling to the maintenance of genome stability.

FANCD2 influences replication fork processes and genome stability in response to clustered DSBs.

Fanconi Anemia (FA) is a cancer predisposition syndrome and the factors defective in FA are involved in DNA replication, DNA damage repair and tumor suppression. Here, we show that FANCD2 is critical for genome stability maintenance in response to high-linear energy transfer (LET) radiation. We found that FANCD2 is monoubiquitinated and recruited to the sites of clustered DNA double-stranded breaks (DSBs) specifically in S/G2 cells after high-LET radiation. Further, FANCD2 facilitated the repair of clustered DSBs in S/G2 cells and proper progression of S-phase. Furthermore, lack of FANCD2 led to a reduced rate of replication fork progression and elevated levels of both replication fork stalling and new origin firing in response to high-LET radiation. Mechanistically, FANCD2 is required for correct recruitment of RPA2 and Rad51 to the sites of clustered DSBs and that is critical for proper processing of clustered DSBs. Significantly, FANCD2-decifient cells exhibited defective chromosome segregation, elevated levels of chromosomal aberrations, and anchorage-independent growth in response to high-LET radiation. These findings establish FANCD2 as a key factor in genome stability maintenance in response to high-LET radiation and as a promising target to improve cancer therapy.

The major DNA repair pathway after both proton and carbon-ion radiation is NHEJ, but the HR pathway is more relevant in carbon ions.

The purpose of this study was to identify the roles of non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways in repairing DNA double-strand breaks (DSBs) induced by exposure to high-energy protons and carbon ions (C ions) versus gamma rays in Chinese hamster cells. Two Chinese hamster cell lines, ovary AA8 and lung fibroblast V79, as well as various mutant sublines lacking DNA-PKcs (V3), X-ray repair cross-complementing protein-4 [XRCC4 (XR1), XRCC3 (irs1SF) and XRCC2 (irs1)] were exposed to gamma rays ((137)Cs), protons (200 MeV; 2.2 keV/µm) and C ions (290 MeV; 50 keV/µm). V3 and XR1 cells lack the NHEJ pathway, whereas irs1 and irs1SF cells lack the HR pathway. After each exposure, survival was measured using a clonogenic survival assay, in situ DSB induction was evaluated by immunocytochemical analysis of histone H2AX phosphorylation at serine 139 (γ-H2AX foci) and chromosome aberrations were examined using solid staining. The findings from this study showed that clonogenic survival clearly depended on the NHEJ and HR pathway statuses, and that the DNA-PKcs(-/-) cells (V3) were the most sensitive to all radiation types. While protons and γ rays yielded almost the same biological effects, C-ion exposure greatly enhanced the sensitivity of wild-type and HR-deficient cells. However, no significant enhancement of sensitivity in cell killing was seen after C-ion irradiation of NHEJ deficient cells. Decreases in the number of γ-H2AX foci after irradiation occurred more slowly in the NHEJ deficient cells. In particular, V3 cells had the highest number of residual γ-H2AX foci at 24 h after C-ion irradiation. Chromosomal aberrations were significantly higher in both the NHEJ- and HR-deficient cell lines than in wild-type cell lines in response to all radiation types. Protons and gamma rays induced the same aberration levels in each cell line, whereas C ions introduced higher but not significantly different aberration levels. Our results suggest that the NHEJ pathway plays an important role in repairing DSBs induced by both clinical proton and C-ion beams. Furthermore, in C ions the HR pathway appears to be involved in the repair of DSBs to a greater extent compared to gamma rays and protons.