Complex DNA Double-strand Breaks Research Articles

GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity.

DNA double-strand break (DSB) induction and repair are important events for determining cell survival and the outcome of cancer radiotherapy. The DNA-dependent protein kinase (DNA-PK) complex functions at the apex of DSBs repair, and its assembly and activity are strictly regulated by post-translation modifications (PTMs)-associated interactions. However, the PTMs of the catalytic subunit DNA-PKcs and how they affect DNA-PKcs's functions are not fully understood. Mass spectrometry analyses were performed to identify the crotonylation sites of DNA-PKcs in response to γ-ray irradiation. Co-immunoprecipitation (Co-IP), western blotting, in vitro crotonylation assays, laser microirradiation assays, in vitro DNA binding assays, in vitro DNA-PK assembly assays and IF assays were employed to confirm the crotonylation, identify the crotonylase and decrotonylase, and elucidate how crotonylation regulates the activity and function of DNA-PKcs. Subcutaneous xenografts of human HeLa GCN5 WT or HeLa GCN5 siRNA cells in BALB/c nude mice were generated and utilized to assess tumor proliferation in vivo after radiotherapy. Here, we reveal that K525 is an important site of DNA-PKcs for crotonylation, and whose level is sharply increased by irradiation. The histone acetyltransferase GCN5 functions as the crotonylase for K525-Kcr, while HDAC3 serves as its dedicated decrotonylase. K525 crotonylation enhances DNA binding activity of DNA-PKcs, and facilitates assembly of the DNA-PK complex. Furthermore, GCN5-mediated K525 crotonylation is indispensable for DNA-PKcs autophosphorylation and the repair of double-strand breaks in the NHEJ pathway. GCN5 suppression significantly sensitizes xenograft tumors of mice to radiotherapy. Our study defines K525 crotonylation of DNA-PKcs is important for the DNA-PK complex assembly and DSBs repair activity via NHEJ pathway. Targeting GCN5-mediated K525 Kcr of DNA-PKcs may be a promising therapeutic strategy for improving the outcome of cancer radiotherapy.

The order of sequential exposure of U2OS cells to gamma and alpha radiation influences the formation and decay dynamics of NBS1 foci.

DNA double strand breaks (DSBs) are a deleterious form of DNA damage. Densely ionising alpha radiation predominantly induces complex DSBs and sparsely ionising gamma radiation-simple DSBs. We have shown that alphas and gammas, when applied simultaneously, interact in producing a higher DNA damage response (DDR) than predicted by additivity. The mechanisms of the interaction remain obscure. The present study aimed at testing whether the sequence of exposure to alphas and gammas has an impact on the DDR, visualised by live NBS1-GFP (green fluorescent protein) focus dynamics in U2OS cells. Focus formation, decay, intensity and mobility were analysed up to 5 h post exposure. Focus frequencies directly after sequential alpha → gamma and gamma → alpha exposure were similar to gamma alone, but gamma → alpha foci quickly declined below the expected values. Focus intensities and areas following alpha alone and alpha → gamma were larger than after gamma alone and gamma → alpha. Focus movement was most strongly attenuated by alpha → gamma. Overall, sequential alpha → gamma exposure induced the strongest change in characteristics and dynamics of NBS1-GFP foci. Possible explanation is that activation of the DDR is stronger when alpha-induced DNA damage precedes gamma-induced DNA damage.

DNA polymerase θ-mediated repair of high LET radiation-induced complex DNA double-strand breaks.

DNA polymerase θ (POLQ) is a unique DNA polymerase that is able to perform microhomology-mediated end-joining as well as translesion synthesis (TLS) across an abasic (AP) site and thymine glycol (Tg). However, the biological significance of the TLS activity is currently unknown. Herein we provide evidence that the TLS activity of POLQ plays a critical role in repairing complex DNA double-strand breaks (DSBs) induced by high linear energy transfer (LET) radiation. Radiotherapy with high LET radiation such as carbon ions leads to more deleterious biological effects than corresponding doses of low LET radiation such as X-rays. High LET-induced DSBs are considered to be complex, carrying additional DNA damage such as AP site and Tg in close proximity to the DSB sites. However, it is not clearly understood how complex DSBs are processed in mammalian cells. We demonstrated that genetic disruption of POLQ results in an increase of chromatid breaks and enhanced cellular sensitivity following treatment with high LET radiation. At the biochemical level, POLQ was able to bypass an AP site and Tg during end-joining and was able to anneal two single-stranded DNA tails when DNA lesions were located outside the microhomology. This study offers evidence that POLQ is directly involved in the repair of complex DSBs.

An Analytical Method for Quantifying the Yields of DNA Double-Strand Breaks Coupled with Strand Breaks by γ-H2AX Focus Formation Assay Based on Track-Structure Simulation.

Complex DNA double-strand break (DSB), which is defined as a DSB coupled with additional strand breaks within 10 bp in this study, induced after ionizing radiation or X-rays, is recognized as fatal damage which can induce cell death with a certain probability. In general, a DSB site inside the nucleus of live cells can be experimentally detected using the γ-H2AX focus formation assay. DSB complexity is believed to be detected by analyzing the focus size using such an assay. However, the relationship between focus size and DSB complexity remains uncertain. In this study, using Monte Carlo (MC) track-structure simulation codes, i.e., an in-house WLTrack code and a Particle and Heavy Ion Transport code System (PHITS), we developed an analytical method for qualifying the DSB complexity induced by photon irradiation from the microscopic image of γ-H2AX foci. First, assuming that events (i.e., ionization and excitation) potentially induce DNA strand breaks, we scored the number of events in a water cube (5.03 × 5.03 × 5.03 nm3) along electron tracks. Second, we obtained the relationship between the number of events and the foci size experimentally measured by the γ-H2AX focus formation assay. Third, using this relationship, we evaluated the degree of DSB complexity induced after photon irradiation for various X-ray spectra using the foci size, and the experimental DSB complexity was compared to the results estimated by the well-verified DNA damage estimation model in the PHITS code. The number of events in a water cube was found to be proportional to foci size, suggesting that the number of events intrinsically related to DSB complexity at the DNA scale. The developed method was applicable to focus data measured for various X-ray spectral situations (i.e., diagnostic kV X-rays and therapeutic MV X-rays). This method would contribute to a precise understanding of the early biological impacts of photon irradiation by means of the γ-H2AX focus formation assay.

The Ubiquitin Ligase RNF138 Cooperates with CtIP to Stimulate Resection of Complex DNA Double-Strand Breaks in Human G1-Phase Cells.

DNA double-strand breaks (DSBs) represent the molecular origin of ionizing-radiation inflicted biological effects. An increase in the ionization density causes more complex, clustered DSBs that can be processed by resection also in G1 phase, where repair of resected DSBs is considered erroneous and may contribute to the increased biological effectiveness of heavy ions in radiotherapy. To investigate the resection regulation of complex DSBs, we exposed G1 cells depleted for different candidate factors to heavy ions or α-particle radiation. Immunofluorescence microscopy was used to monitor the resection marker RPA, the DSB marker γH2AX and the cell-cycle markers CENP-F and geminin. The Fucci system allowed to select G1 cells, cell survival was measured by clonogenic assay. We show that in G1 phase the ubiquitin ligase RNF138 functions in resection regulation. RNF138 ubiquitinates the resection factor CtIP in a radiation-dependent manner to allow its DSB recruitment in G1 cells. At complex DSBs, RNF138′s participation becomes more relevant, consistent with the observation that also resection is more frequent at these DSBs. Furthermore, deficiency of RNF138 affects both DSB repair and cell survival upon induction of complex DSBs. We conclude that RNF138 is a regulator of resection that is influenced by DSB complexity and can affect the quality of DSB repair in G1 cells.

Formation of clustered DNA damage in vivo upon irradiation with ionizing radiation: Visualization and analysis with atomic force microscopy

SignificanceDNA damage causes loss of or alterations in genetic information, resulting in cell death or mutations. Ionizing radiations produce local, multiple DNA damage sites called clustered DNA damage. In this study, a complete protocol was established to analyze the damage complexity of clustered DNA damage, wherein damage-containing genomic DNA fragments were selectively concentrated via pulldown, and clustered DNA damage was visualized by atomic force microscopy. It was found that X-rays and Fe ion beams caused clustered DNA damage. Fe ion beams also produced clustered DNA damage with high complexity. Fe ion beam-induced complex DNA double-strand breaks (DSBs) containing one or more base lesion(s) near the DSB end were refractory to repair, implying their lethal effects.

DNA Damage Clustering after Ionizing Radiation and Consequences in the Processing of Chromatin Breaks

Charged-particle radiotherapy (CPRT) utilizing low and high linear energy transfer (low-/high-LET) ionizing radiation (IR) is a promising cancer treatment modality having unique physical energy deposition properties. CPRT enables focused delivery of a desired dose to the tumor, thus achieving a better tumor control and reduced normal tissue toxicity. It increases the overall radiation tolerance and the chances of survival for the patient. Further improvements in CPRT are expected from a better understanding of the mechanisms governing the biological effects of IR and their dependence on LET. There is increasing evidence that high-LET IR induces more complex and even clustered DNA double-strand breaks (DSBs) that are extremely consequential to cellular homeostasis, and which represent a considerable threat to genomic integrity. However, from the perspective of cancer management, the same DSB characteristics underpin the expected therapeutic benefit and are central to the rationale guiding current efforts for increased implementation of heavy ions (HI) in radiotherapy. Here, we review the specific cellular DNA damage responses (DDR) elicited by high-LET IR and compare them to those of low-LET IR. We emphasize differences in the forms of DSBs induced and their impact on DDR. Moreover, we analyze how the distinct initial forms of DSBs modulate the interplay between DSB repair pathways through the activation of DNA end resection. We postulate that at complex DSBs and DSB clusters, increased DNA end resection orchestrates an increased engagement of resection-dependent repair pathways. Furthermore, we summarize evidence that after exposure to high-LET IR, error-prone processes outcompete high fidelity homologous recombination (HR) through mechanisms that remain to be elucidated. Finally, we review the high-LET dependence of specific DDR-related post-translational modifications and the induction of apoptosis in cancer cells. We believe that in-depth characterization of the biological effects that are specific to high-LET IR will help to establish predictive and prognostic signatures for use in future individualized therapeutic strategies, and will enhance the prospects for the development of effective countermeasures for improved radiation protection during space travel.

Immediate-Early, Early, and Late Responses to DNA Double Stranded Breaks.

Loss or rearrangement of genetic information can result from incorrect responses to DNA double strand breaks (DSBs). The cellular responses to DSBs encompass a range of highly coordinated events designed to detect and respond appropriately to the damage, thereby preserving genomic integrity. In analogy with events occurring during viral infection, we appropriate the terms Immediate-Early, Early, and Late to describe the pre-repair responses to DSBs. A distinguishing feature of the Immediate-Early response is that the large protein condensates that form during the Early and Late response and are resolved upon repair, termed foci, are not visible. The Immediate-Early response encompasses initial lesion sensing, involving poly (ADP-ribose) polymerases (PARPs), KU70/80, and MRN, as well as rapid repair by so-called ‘fast-kinetic’ canonical non-homologous end joining (cNHEJ). Initial binding of PARPs and the KU70/80 complex to breaks appears to be mutually exclusive at easily ligatable DSBs that are repaired efficiently by fast-kinetic cNHEJ; a process that is PARP-, ATM-, 53BP1-, Artemis-, and resection-independent. However, at more complex breaks requiring processing, the Immediate-Early response involving PARPs and the ensuing highly dynamic PARylation (polyADP ribosylation) of many substrates may aid recruitment of both KU70/80 and MRN to DSBs. Complex DSBs rely upon the Early response, largely defined by ATM-dependent focal recruitment of many signalling molecules into large condensates, and regulated by complex chromatin dynamics. Finally, the Late response integrates information from cell cycle phase, chromatin context, and type of DSB to determine appropriate pathway choice. Critical to pathway choice is the recruitment of p53 binding protein 1 (53BP1) and breast cancer associated 1 (BRCA1). However, additional factors recruited throughout the DSB response also impact upon pathway choice, although these remain to be fully characterised. The Late response somehow channels DSBs into the appropriate high-fidelity repair pathway, typically either ‘slow-kinetic’ cNHEJ or homologous recombination (HR). Loss of specific components of the DSB repair machinery results in cells utilising remaining factors to effect repair, but often at the cost of increased mutagenesis. Here we discuss the complex regulation of the Immediate-Early, Early, and Late responses to DSBs proceeding repair itself.

Blm helicase facilitates rapid replication of repetitive DNA sequences in early Drosophila development.

The absence of functional BLM DNA helicase, a member of the RecQ family of helicases, is responsible for the rare human disorder Bloom Syndrome, which results in developmental abnormalities, DNA repair defects, genomic instability, and a predisposition to cancer. In Drosophila melanogaster, the orthologous Blm protein is essential during early development when the embryo is under the control of maternal gene products. We show that lack of functional maternal Blm during the syncytial cell cycles of Drosophila embryonic development results in severe nuclear defects and lethality. Amongst the small fraction of embryos from Blm mutant mothers that survive to adulthood, a prominent sex-bias favors the class that inherits less repetitive DNA content, which serves as an endogenous source of replication stress. This selection against repetitive DNA content reflects a role for Blm in facilitating replication through repetitive sequences during the rapid S-phases of syncytial cell cycles. During these syncytial cycles, Blm is not required for complex DNA double-strand break repair; however, the progeny sex-bias resulting from the absence of maternal Blm is exacerbated by repetitive DNA sequences and by the slowing of replication fork progression, suggesting that the essential role for Blm during this stage is to manage replication fork stress brought about by impediments to fork progression. Additionally, our data suggest that Blm is only required to manage this replication stress during embryonic development, and likely only during the early, rapid syncytial cell cycles, and not at later developmental stages. These results provide novel insights into Blm function throughout development.

Towards the characterization of neutron carcinogenesis through direct action simulations of clustered DNA damage

Neutron exposure poses a unique radiation protection concern because neutrons have a large, energy-dependent relative biological effectiveness (RBE) for stochastic effects. Recent computational studies on the microdosimetric properties of neutron dose deposition have implicated clustered DNA damage as a likely contributor to this marked energy dependence. So far, publications have focused solely on neutron RBE for inducing clusters of DNA damage containing two or more DNA double strand breaks (DSBs). In this study, we have conducted a novel assessment of neutron RBE for inducing all types of clustered DNA damage that contain two or more lesions, stratified by whether the clusters contain DSBs (complex DSB clusters) or not (non-DSB clusters). This assessment was conducted for eighteen initial neutron energies between 1 eV and 10 MeV as well as a reference radiation of 250 keV x-rays. We also examined the energy dependence of cluster length and cluster complexity because these factors are believed to impact the DNA repair process. To carry out our investigation, we developed a user-friendly TOPAS-nBio application that includes a custom nuclear DNA model and a novel algorithm for recording clustered DNA damage. We found that neutron RBE for inducing complex DSB clusters exhibited similar energy dependence to the canonical neutron RBE for stochastic radiobiological effects, at multiple depths in human tissue. Qualitatively similar results were obtained for non-DSB clusters, although the quantitative agreement was lower. Additionally we identified a significant neutron energy dependence in the average length and complexity of clustered lesions. These results support the idea that many types of clustered DNA damage contribute to the energy dependence of neutron RBE for stochastic radiobiological effects and imply that the size and constituent lesions of individual clusters should be taken into account when modeling DNA repair. Our results were qualitatively consistent for (i) multiple radiation doses (including a low-dose 0.1 Gy irradiation), (ii) variations in the maximal lesion separation distance used to define a cluster, and (iii) two distinct collections of physics models used to govern particle transport. Our complete TOPAS-nBio application has been released under an open-source license to enable others to independently validate our work and to expand upon it.

Elucidation of the Clustered Nano-Architecture of Radiation-Induced DNA Damage Sites and Surrounding Chromatin in Cancer Cells: A Single Molecule Localization Microscopy Approach.

In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a “double-edged sword”—it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.

Inhibition of ATM Induces Hypersensitivity to Proton Irradiation by Upregulating Toxic End Joining.

Proton Bragg peak irradiation has a higher ionizing density than conventional photon irradiation or the entrance of the proton beam profile. Whether targeting the DNA damage response (DDR) could enhance vulnerability to the distinct pattern of damage induced by proton Bragg peak irradiation is currently unknown. Here, we performed genetic or pharmacologic manipulation of key DDR elements and evaluated DNA damage signaling, DNA repair, and tumor control in cell lines and xenografts treated with the same physical dose across a radiotherapy linear energy transfer spectrum. Radiotherapy consisted of 6 MV photons and the entrance beam or Bragg peak of a 76.8 MeV spot scanning proton beam. More complex DNA double-strand breaks (DSB) induced by Bragg peak proton irradiation preferentially underwent resection and engaged homologous recombination (HR) machinery. Unexpectedly, the ataxia-telangiectasia mutated (ATM) inhibitor, AZD0156, but not an inhibitor of ATM and Rad3-related, rendered cells hypersensitive to more densely ionizing proton Bragg peak irradiation. ATM inhibition blocked resection and shunted more DSBs to processing by toxic ligation through nonhomologous end-joining, whereas loss of DNA ligation via XRCC4 or Lig4 knockdown rescued resection and abolished the enhanced Bragg peak cell killing. Proton Bragg peak monotherapy selectively sensitized cell lines and tumor xenografts with inherent HR defects, and the repair defect induced by ATM inhibitor coadministration showed enhanced efficacy in HR-proficient models. In summary, inherent defects in HR or administration of an ATM inhibitor in HR-proficient tumors selectively enhances the relative biological effectiveness of proton Bragg peak irradiation. SIGNIFICANCE: Coadministration of an ATM inhibitor rewires DNA repair machinery to render cancer cells uniquely hypersensitive to DNA damage induced by the proton Bragg peak, which is characterized by higher density ionization.See related commentary by Nickoloff, p. 3156.

Enhanced Cell Inactivation and Double-Strand Break Induction in V79 Chinese Hamster Cells by Monochromatic X-Rays at Phosphorus K-Shell Absorption Peak

The cell inactivation and DNA double-strand break (DSB) induction by K-shell ionization of phosphorus atoms and Auger electrons were investigated. Monochromatic X-rays of on and below the phosphorus K-shell absorption peak, 2.153 keV and 2.147 keV were exposed to Chinese hamster lung fibroblast V79 cells. Survival fractions were plotted against exposure, Ψ [nC/kg] and the linear-quadratic model was adapted to estimate the parameters, α and β, of the survival curves. DSB induction rate [DSB/cell/Ψ] was estimated from the measured fractions of induced DNA fragments below 4.6 Mbp (Find(k < 4.6)), which were determined using pulse field gel electrophoresis. As results, cell inactivation and DSB induction rate of on the peak were significantly higher compared to that of the below. However, when converting Ψ to absorbed dose (Gy) of cell nucleus, the enhanced effect was only observed for parameter α, and not for a survival dose (Gy) of 37%, 10%, and 1% nor for a DSB induction rate. Our findings indicate that enhancement of cell inactivation and DSB induction were due to the additional dose delivered to the DNA and more complex DSB lesions were induced due to the release of phosphorus K-shell photoelectrons and Auger electrons.

Differential Repair Protein Recruitment at Sites of Clustered and Isolated DNA Double-Strand Breaks Produced by High-Energy Heavy Ions

DNA double-strand break (DSB) repair is crucial to maintain genomic stability. The fidelity of the repair depends on the complexity of the lesion, with clustered DSBs being more difficult to repair than isolated breaks. Using live cell imaging of heavy ion tracks produced at a high-energy particle accelerator we visualised simultaneously the recruitment of different proteins at individual sites of complex and simple DSBs in human cells. NBS1 and 53BP1 were recruited in a few seconds to complex DSBs, but in 40% of the isolated DSBs the recruitment was delayed approximately 5 min. Using base excision repair (BER) inhibitors we demonstrate that some simple DSBs are generated by enzymatic processing of base damage, while BER did not affect the complex DSBs. The results show that DSB processing and repair kinetics are dependent on the complexity of the breaks and can be different even for the same clastogenic agent.

A Stochastic Model of DNA Double-Strand Breaks Repair Throughout the Cell Cycle.

Cell cycle phase is a decisive factor in determining the repair pathway of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ) or homologous recombination (HR). Recent experimental studies revealed that 53BP1 and BRCA1 are the key mediators of the DNA damage response (DDR) with antagonizing roles in choosing the appropriate DSB repair pathway in G1, S, and G2 phases. Here, we present a stochastic model of biochemical kinetics involved in detecting and repairing DNA DSBs induced by ionizing radiation during the cell cycle progression. A three-dimensional stochastic process is defined to monitor the cell cycle phase and DSBs repair at times after irradiation. To estimate the model parameters, a Metropolis Monte Carlo method is applied to perform maximum likelihood estimation utilizing the kinetics of γ-H2AX and RAD51 foci formation in G1, S, and G2 phases. The recruitment of DSB repair proteins is verified by comparing our model predictions with the corresponding experimental data on human cells after exposure to X and γ-radiation. Furthermore, the interaction between 53BP1 and BRCA1 is simulated for G1 and S/G2 phases determining the competition between NHEJ and HR pathways in repairing induced DSBs throughout the cell cycle. In accordance with recent biological data, the numerical results demonstrate that the maximum proportion of HR occurs in S phase cells and the high level of NHEJ takes place in G1 and G2 phases. Moreover, the stochastic realizations of the total yield of simple and complex DSBs ligation are compared for G1 and S/G2 damaged cells. Finally, the proposed stochastic model is validated when DSBs induced by different particle radiation such as iron, silicon, oxygen, proton, and carbon.

Stochastic multicellular modeling of x-ray irradiation, DNA damage induction, DNA free-end misrejoining and cell death

The repair or misrepair of DNA double-strand breaks (DSBs) largely determines whether a cell will survive radiation insult or die. A new computational model of multicellular, track structure-based and pO2-dependent radiation-induced cell death was developed and used to investigate the contribution to cell killing by the mechanism of DNA free-end misrejoining for low-LET radiation. A simulated tumor of 1224 squamous cells was irradiated with 6 MV x-rays using the Monte Carlo toolkit Geant4 with low-energy Geant4-DNA physics and chemistry modules up to a uniform dose of 1 Gy. DNA damage including DSBs were simulated from ionizations, excitations and hydroxyl radical interactions along track segments through cell nuclei, with a higher cellular pO2 enhancing the conversion of DNA radicals to strand breaks. DNA free-ends produced by complex DSBs (cDSBs) were able to misrejoin and produce exchange-type chromosome aberrations, some of which were asymmetric and lethal. A sensitivity analysis was performed and conditions of full oxia and anoxia were simulated. The linear component of cell killing from misrejoining was consistently small compared to values in the literature for the linear component of cell killing for head and neck squamous cell carcinoma (HNSCC). This indicated that misrejoinings involving DSBs from the same x-ray (including all associated secondary electrons) were rare and that other mechanisms (e.g. unrejoined ends) may be important. Ignoring the contribution by the indirect effect toward DNA damage caused the DSB yield to drop to a third of its original value and the cDSB yield to drop to a tenth of its original value. Track structure-based cell killing was simulated in all 135306 viable cells of a 1 mm3 hypoxic HNSCC tumor for a uniform dose of 1 Gy.

Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

ABSTRACTNucleoli have attracted interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription and nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and the presence (or not) of DSBs outside the nucleoli. To address the controversy, we targeted nucleoli with carbon ions at the ion microbeam SNAKE. Localized ion irradiation with 1–100 carbon ions per point (about 0.3–30 Gy per nucleus) did not lead to overall reduced ribonucleotide incorporation in the targeted nucleolus or other nucleoli of the same cell. However, both 5-ethynyluridine incorporation and Parp1 protein levels were locally decreased at the damaged nucleolar chromatin regions marked by γH2AX, suggesting localized inhibition of rRNA transcription. This locally restricted transcriptional inhibition was not accompanied by nucleolar segregation, a structural reorganisation observed after inhibition of rRNA transcription by treatment with actinomycin D or UV irradiation. The presented data indicate that even multiple complex DSBs do not lead to a pan-nucleolar response if they affect only a subnucleolar region.

Radiation Track Structure: How the Spatial Distribution of Energy Deposition Drives Biological Response

Ionising radiation is incredibly effective at causing biological effects. This is due to the unique way energy is deposited along highly structured tracks of ionisation and excitation events, which results in correlation with sites of DNA damage from the nanometre to the micrometre scale. Correlation of these events along the track on the nanometre scale results in clustered damage, which not only results in the formation of DNA double-strand breaks (DSB), but also more difficult to repair complex DSB, which include additional damage within a few base pairs. The track structure varies significantly with radiation quality and the increase in relative biological effectiveness observed with increasing linear energy transfer in part corresponds to an increase in the probability and complexity of clustered DNA damage produced. Likewise, correlation over larger scales, associated with packing of DNA and associated chromosomes within the cell nucleus, can also have a major impact on the biological response. The proximity of the correlated damage along the track increases the probability of miss-repair through pairwise interactions resulting in an increase in probability and complexity of DNA fragments/deletions, mutations and chromosomal rearrangements. Understanding the mechanisms underlying the biological effectiveness of ionising radiation can provide an important insight into ways of increasing the efficacy of radiotherapy, as well as the risks associated with exposure. This requires a multi-scale approach for modelling, not only considering the physics of the track structure from the millimetre scale down to the nanometre scale, but also the structural packing of the DNA within the nucleus, the resulting chemistry in the context of the highly reactive environment of the nucleus, together with the subsequent biological response.

Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2'-guanosine.

DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2'-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

Synergistic Effect of a Mesothelin-Targeted 227Th Conjugate in Combination with DNA Damage Response Inhibitors in Ovarian Cancer Xenograft Models.

Targeted 227Th conjugates (TTCs) represent a new class of therapeutic radiopharmaceuticals for targeted α-therapy. They comprise the α-emitter 227Th complexed to a 3,2-hydroxypyridinone chelator conjugated to a tumor-targeting monoclonal antibody. The high energy and short range of the α-particles induce antitumor activity, driven by the induction of complex DNA double-strand breaks. We hypothesized that blocking the DNA damage response (DDR) pathway should further sensitize cancer cells by inhibiting DNA repair, thereby increasing the response to TTCs. Methods: This article reports the evaluation of the mesothelin (MSLN)-TTC conjugate (BAY 2287411) in combination with several DDR inhibitors, each of them blocking different DDR pathway enzymes. MSLN is a validated cancer target known to be overexpressed in mesothelioma, ovarian, lung, breast, and pancreatic cancer, with low expression in normal tissue. In vitro cytotoxicity experiments were performed on cancer cell lines by combining the MSLN-TTC with inhibitors of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related (ATR), DNA-dependent protein kinase, and poly[adenosine diphosphate ribose] polymerase (PARP) 1/2. Further, we evaluated the antitumor efficacy of the MSLN-TTC in combination with DDR inhibitors in human ovarian cancer xenograft models. Results: Synergistic activity was observed in vitro for all tested inhibitors (inhibitors are denoted herein by the suffix “i”) when combined with MSLN-TTC. ATRi and PARPi appeared to induce the strongest increase in potency. Further, in vivo antitumor efficacy of the MSLN-TTC in combination with ATRi or PARPi was investigated in the OVCAR-3 and OVCAR-8 xenograft models in nude mice, demonstrating synergistic antitumor activity for the ATRi combination at doses demonstrated to be nonefficacious when administered as monotherapy. Conclusion: The presented data support the mechanism-based rationale for combining the MSLN-TTC with DDR inhibitors as new treatment strategies in MSLN-positive ovarian cancer.