DNA Damage Foci on Metaphase Chromosomes.

After DNAs are damaged, DNA repair proteins accumulate and are activated at the DNA damaged site. These accumulated proteins are visualized as foci by fluorescent immunocytochemistry technique. This allows the DNA damage responses in interphase nuclei to be detected; it was earlier times difficult to analyze DNA damage in situ. In order to analyze DNA damage in interphase cells, either DNA is extracted to assay breaks biochemically, or premature chromosome condensation is conducted to observe as chromatin breaks. Although DNA damage-induced foci are typically analyzed in interphase cells, these foci can be also visualized on mitotic chromosomes. The foci where the repair proteins accumulate at the damage site is observed as mitotic chromosome break site. Since mitotic cells attach loosely or not attached to cell culture vessels, it is difficult to analyze foci on chromosomes in culture vessels under a microscope, so metaphase chromosome spread must be prepared for accurate analysis. The cytocentrifuge system is an ideal method to adhere mitotic cells to microscope slides for the fluorescent immunocytochemistry. This chapter introduces cytocentrifuge method to prepare metaphase spread for DNA damage foci analysis.

Dose response to methylating agents in the γH2AX, SCE and colony formation assays: Effect of MGMT and MPG overexpression.

Cells have developed diverse protective mechanisms that enable them to tolerate low doses of genotoxic compounds. DNA repair processes attenuate the mutagenic and carcinogenic effects of alkylating agents, and multiple studies indicate a key role of specific DNA repair factors and pathways in establishing non-linear dose response relationships. Using an overexpression approach, we investigated the impact of O6-methylguanine-DNA-methyltransferase (MGMT), which repairs O6-methylguanine (O6MeG) in a damage reversal reaction, and N-methylpurine-DNA glycosylase (MPG), which acts as an apical enzyme in the BER pathway, on the DNA damage response to the alkylating agents MNNG and MMS. Our data indicate a clear protective effect of MGMT against MNNG-induced nuclear γH2AX foci formation, sister chromatid exchanges (SCE) and cytotoxicity, as determined in the colony formation assay. MGMT protected with similar efficiency against MMS-induced cytotoxicity and γH2AX foci formation, but suppressed SCE induction only weakly, which indicates that recombination events induced by MMS result from other lesions than O6MeG. In contrast, overexpression of MPG had only a very mild protective effect on the cellular defense against MMS and MNNG. Collectively, our data indicate that overexpression of MGMT results in non-linear DNA damage responses to O6MeG inducers. In contrast, MPG overexpression has only minor impact on the DNA damage response to alkylating drugs, indicating that other downstream enzymes in the BER pathway are limiting.

Assessing kinetics and recruitment of DNA repair factors using high content screens.

Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library ("ChromORFeome" library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.

The evolving complexity of DNA damage foci: RNA, condensates and chromatin in DNA double-strand break repair.

Formation of biomolecular condensates is increasingly recognized as a mechanism employed by cells to deal with stress and to optimize enzymatic reactions. Recent studies have characterized several DNA repair foci as phase-separated condensates, behaving like liquid droplets. Concomitantly, the apparent importance of long non-coding RNAs and RNA-binding proteins for the repair of double-strand breaks has raised many questions about their exact contribution to the repair process. Here we discuss how RNA molecules can participate in condensate formation and how RNA-binding proteins can act as molecular scaffolds. We furthermore summarize our current knowledge about how properties of condensates can influence the choice of repair pathway (homologous recombination or non-homologous end joining) and identify the open questions in this field of emerging importance.

Analysis of the Contribution of NF-κB in the Regulation of Chemotherapy-Induced Cell Senescence by Establishing a Tetracycline-Regulated Cell System.

Therapy-induced senescence (TIS or therapy-induced premature senescence) is a key cellular program triggered in the course of cancer radiotherapy and chemotherapy with genotoxic drugs, both in cancer cells and in normal cells, whose activation critically affects the outcome of cancer therapy. Drug-induced senescent cells undergo a permanent cell cycle arrest, acquire distinctive morphological and biochemical alterations, and an enhanced secretory ability, referred to as senescence-associated secretory phenotype (SASP). The transcription factor NF-κB acts as a master regulator of the SASP, driving the expression of senescence-associated secretome components.Here we describe protocols for the establishment of a tetracycline-regulated cell system for the investigation of the role of NF-κB in TIS. We also describe protocols routinely used in our laboratory, to investigate TIS in this Tet-On inducible expression system. Finally, we describe techniques for the validation of TIS induction.

Proton Irradiation Increases the Necessity for Homologous Recombination Repair Along with the Indispensability of Non-Homologous End Joining.

Technical improvements in clinical radiotherapy for maximizing cytotoxicity to the tumor while limiting negative impact on co-irradiated healthy tissues include the increasing use of particle therapy (e.g., proton therapy) worldwide. Yet potential differences in the biology of DNA damage induction and repair between irradiation with X-ray photons and protons remain elusive. We compared the differences in DNA double strand break (DSB) repair and survival of cells compromised in non-homologous end joining (NHEJ), homologous recombination repair (HRR) or both, after irradiation with an equal dose of X-ray photons, entrance plateau (EP) protons, and mid spread-out Bragg peak (SOBP) protons. We used super-resolution microscopy to investigate potential differences in spatial distribution of DNA damage foci upon irradiation. While DNA damage foci were equally distributed throughout the nucleus after X-ray photon irradiation, we observed more clustered DNA damage foci upon proton irradiation. Furthermore, deficiency in essential NHEJ proteins delayed DNA repair kinetics and sensitized cells to both, X-ray photon and proton irradiation, whereas deficiency in HRR proteins sensitized cells only to proton irradiation. We assume that NHEJ is indispensable for processing DNA DSB independent of the irradiation source, whereas the importance of HRR rises with increasing energy of applied irradiation.

DNA damage accumulation during fractionated low-dose radiation compromises hippocampal neurogenesis.

BACKGROUND AND PURPOSE: High-precision radiotherapy is an effective treatment modality for tumors. Intensity-modulated radiotherapy techniques permit close shaping of high doses to tumors, however healthy organs outside the target volume are repeatedly exposed to low-dose radiation (LDR). The inherent vulnerability of hippocampal neurogenesis is likely the determining factor in radiation-induced neurocognitive dysfunctions. Using preclinical in-vivo models with daily LDR we attempted to precisely define the pathophysiology of radiation-induced neurotoxicity. MATERIAL AND METHODS: Genetically defined mouse strains with varying DNA repair capacities were exposed to fractionated LDR (5×/10×/15×/20×0.1 Gy) and dentate gyri from juvenile and adult mice were analyzed 72 h after last exposure and 1, 3, 6 months after 20 × 0.1 Gy. To examine the impact of LDR on neurogenesis, persistent DNA damage was assessed by quantifying 53BP1-foci within hippocampal neurons. Moreover, subpopulations of neuronal stem/progenitor cells were quantified and dendritic arborization of developing neurons were assessed. To unravel molecular mechanisms involved in radiation-induced neurotoxicity, hippocampi were analyzed using mass spectrometry-based proteomics and affected signaling networks were validated by immunoblotting. RESULTS: Radiation-induced DNA damage accumulation leads to progressive decline of hippocampal neurogenesis with decreased numbers of stem/progenitor cells and reduced complexities of dendritic architectures, clearly more pronounced in repair-deficient mice. Proteome analysis revealed substantial changes in neurotrophic signaling, with strong suppression directly after LDR and compensatory upregulation later on to promote functional recovery. CONCLUSION: Hippocampal neurogenesis is highly sensitive to repetitive LDR. Even low doses affect signaling networks within the neurogenic niche and interrupt the dynamic process of generation and maturation of neuronal stem/progenitor cells.

A cmap-enabled gene expression signature-matching approach identifies small-molecule inducers of accelerated cell senescence.

BACKGROUND: Diverse stresses including genotoxic therapy can induce proliferating cancer cells to undergo cellular senescence and take on the characteristic phenotypes of replicative cellular aging. This accelerated or therapy-induced senescence has been alternatively proposed to contribute to therapeutic efficacy or resistance. Toward better understanding this cell state, we sought to define the core transcriptome of accelerated senescence in cancer cells. RESULTS: We examined senescence induced by ionizing irradiation or ectopic overexpression of the stoichiometric cyclin-dependent kinase (CDK) inhibitor p21CIP/WAF1/SDI1 in the human breast cancer cell line MCF7. While radiation produces a strong DNA damage response, ectopic expression of p21 arrests cell cycle progression independently of DNA damage. Both conditions promoted senescence within 5 days. Microarray analysis revealed 378 up- and 391 down-regulated genes that were shared between the two conditions, representing a candidate signature. Systems analysis of the shared differentially expressed genes (DEGs) revealed strong signals for cell cycle control and DNA damage response pathways and predicted multiple upstream regulators previously linked to senescence. Querying the shared DEGs against the Connectivity Map (cmap) database of transcriptional responses to small molecules yielded 20 compounds that induce a similar gene expression pattern in MCF7 cells. Of 16 agents evaluated, six induced senescence on their own. Of these, the selective estrogen receptor degrader fulvestrant and the histone acetyltransferase inhibitor vorinostat did so without causing chromosomal damage. CONCLUSIONS: Using a systems biology approach with experimental validation, we have defined a core gene expression signature for therapy-induced senescence.

Quantifying Senescence-Associated Phenotypes in Primary Multipotent Mesenchymal Stromal Cell Cultures.

Cellular senescence is a tumor suppressor mechanism that removes potentially neoplastic cells from the proliferative pool. Senescent cells naturally accumulate with advancing age; however, excessive/aberrant accumulation of senescent cells can disrupt normal tissue function. Multipotent mesenchymal stromal cells (MSCs), which are actively evaluated as cell-based therapy, can undergo replicative senescence or stress-induced premature senescence. The molecular characterization of MSCs senescence can be useful not only for understanding the clinical correlations between MSCs biology and human age or age-related diseases but also for identifying competent MSCs for therapeutic applications. Because MSCs are involved in regulating the hematopoietic stem cell niche, and MSCs dysfunction has been implicated in age-related diseases, the identification and selective removal of senescent MSC may represent a potential therapeutic target. Cellular senescence is generally defined by senescence-associated (SA) permanent proliferation arrest (SAPA) accompanied by persistent DNA damage response (DDR) signaling emanating from persistent DNA lesions including damaged telomeres. Alongside SA cell cycle arrest and DDR signaling, a plethora of phenotypic hallmarks help define the overall senescent phenotype including a potent SA secretory phenotype (SASP) with many microenvironmental functions. Due to the complexity of the senescence phenotype, no single hallmark is alone capable of identifying senescent MSCs. This protocol highlights strategies to validate MSCs senescence through the measurements of several key SA hallmarks including lysosomal SA Beta-galactosidase activity (SA-ßgal), cell cycle arrest, persistent DDR signaling, and the inflammatory SASP.

Persistent accumulation of unrepaired DNA damage in rat cortical neurons: nuclear organization and ChIP-seq analysis of damaged DNA.

Neurons are highly vulnerable to DNA damage induced by genotoxic agents such as topoisomerase activity, oxidative stress, ionizing radiation (IR) and chemotherapeutic drugs. To avert the detrimental effects of DNA lesions in genome stability, transcription and apoptosis, neurons activate robust DNA repair mechanisms. However, defective DNA repair with accumulation of unrepaired DNA are at the basis of brain ageing and several neurodegenerative diseases. Understanding the mechanisms by which neurons tolerate DNA damage accumulation as well as defining the genomic regions that are more vulnerable to DNA damage or refractory to DNA repair and therefore constitute potential targets in neurodegenerative diseases are essential issues in the field. In this work we investigated the nuclear topography and organization together with the genome-wide distribution of unrepaired DNA in rat cortical neurons 15 days upon IR. About 5% of non-irradiated and 55% of irradiated cells accumulate unrepaired DNA within persistent DNA damage foci (PDDF) of chromatin. These PDDF are featured by persistent activation of DNA damage/repair signaling, lack of transcription and localization in repressive nuclear microenvironments. Interestingly, the chromatin insulator CTCF is concentrated at the PDDF boundaries, likely contributing to isolate unrepaired DNA from intact transcriptionally active chromatin. By confining damaged DNA, PDDF would help preserving genomic integrity and preventing the production of aberrant proteins encoded by damaged genes.ChIP-seq analysis of genome-wide γH2AX distribution revealed a number of genomic regions enriched in γH2AX signal in IR-treated cortical neurons. Some of these regions are in close proximity to genes encoding essential proteins for neuronal functions and human neurodegenerative disorders such as epm2a (Lafora disease), serpini1 (familial encephalopathy with neuroserpin inclusion bodies) and il1rpl1 (mental retardation, X-linked 21). Persistent γH2AX signal close to those regions suggests that nearby genes could be either more vulnerable to DNA damage or more refractory to DNA repair.

Image-Based High Content Screening: Automating the Quantification Process for DNA Damage-Induced Foci.

Visual inspection of cellular activities based on conventional fluorescence microscope is a fundamental tool to study the role of DNA damage response (DDR). In the context of drug discovery where the capture of thousands of images is required across parallel experiments, this presents a challenge to data collection and analysis. Manual scoring is laborious and often reliant on trained personnel to intuit biological meaning through visual reasoning. On the other hand, high content screening combines the automation of microscopy image acquisition and analysis in a single platform to quantify cellular events of interests. The data generated is rapid and accurate, lessening the bias of human interpretation. Herein, this chapter will describe an image-based high content screen approach and the data analysis of Ataxia-Telangiectasia Mutated (ATM) DNA damage-induced foci.

Monitoring DNA Repair Consequences of ATM Signaling Using Simultaneous Fluorescent Readouts.

The repair of deleterious DNA double strand breaks is required to maintain genome integrity. The efficacy in which this occurs relies upon the available machinery and is guided by factors that include cell cycle status, availability of donor template, and the local chromosome structure. Therefore at a single DNA breakpoint there are different outcomes that can occur. The Traffic light reporter (TLR) assay protocol is a dual fluorescent readout that has the ability to monitor simultaneous homologous recombination and non-homologous end joining activity in response to DNA damage. This provides insight to determine the upstream functionality of either pathway mediated through ATM.

DNA Damage Detection by 53BP1: Relationship to Species Longevity.

In order to examine potential differences in genomic stability, we have challenged fibroblasts derived from five different mammalian species of variable longevity with the genotoxic agents, etoposide and neocarzinostatin. We report that cells from longer-lived species exhibit more tumor protein p53 binding protein 1 (53BP1) foci for a given degree of DNA damage relative to shorter-lived species. The presence of a greater number of 53BP1 foci was associated with decreased DNA fragmentation and a lower percentage of cells exhibiting micronuclei. These data suggest that cells from longer-lived species have an enhanced DNA damage response. We propose that the number of 53BP1 foci that form in response to damage reflects the intrinsic capacity of cells to detect and respond to DNA harms.

VRK1 phosphorylates and protects NBS1 from ubiquitination and proteasomal degradation in response to DNA damage.

NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (-/-) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (-/-) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (-/-) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR.

Correlation of Particle Traversals with Clonogenic Survival Using Cell-Fluorescent Ion Track Hybrid Detector.

Development of novel approaches linking the physical characteristics of particles with biological responses are of high relevance for the field of particle therapy. In radiobiology, the clonogenic survival of cells is considered the gold standard assay for the assessment of cellular sensitivity to ionizing radiation. Toward further development of next generation biodosimeters in particle therapy, cell-fluorescent ion track hybrid detector (Cell-FIT-HD) was recently engineered by our group and successfully employed to study physical particle track information in correlation with irradiation-induced DNA damage in cell nuclei. In this work, we investigated the feasibility of Cell-FIT-HD as a tool to study the effects of clinical beams on cellular clonogenic survival. Tumor cells were grown on the fluorescent nuclear track detector as cell culture, mimicking the standard procedures for clonogenic assay. Cell-FIT-HD was used to detect the spatial distribution of particle tracks within colony-initiating cells. The physical data were associated with radiation-induced foci as surrogates for DNA double-strand breaks, the hallmark of radiation-induced cell lethality. Long-term cell fate was monitored to determine the ability of cells to form colonies. We report the first successful detection of particle traversal within colony-initiating cells at subcellular resolution using Cell-FIT-HD.