DNA Double-strand Break Response Research Articles

Cardiomyocytes, cardiac endothelial cells and fibroblasts contribute to anthracycline-induced cardiac injury through RAS-homologous small GTPases RAC1 and CDC42

The clinical use of the DNA damaging anticancer drug doxorubicin (DOX) is limited by irreversible cardiotoxicity, which depends on the cumulative dose. The RAS-homologous (RHO) small GTPase RAC1 contributes to DOX-induced DNA damage formation and cardiotoxicity. However, the pathophysiological relevance of other RHO GTPases than RAC1 and different cardiac cell types (i.e., cardiomyocytes, non-cardiomyocytes) for DOX-triggered cardiac damage is unclear. Employing diverse in vitro and in vivo models, we comparatively investigated the level of DOX-induced DNA damage in cardiomyocytes versus non-cardiomyocytes (endothelial cells and fibroblasts), in the presence or absence of selected RHO GTPase inhibitors. Non-cardiomyocytes exhibited the highest number of DOX-induced DNA double-strand breaks (DSB), which were efficiently repaired in vitro. By contrast, rather low levels of DSB were formed in cardiomyocytes, which however remained largely unrepaired. Moreover, DOX-induced apoptosis was detected only in non-cardiomyocytes but not in cardiomyocytes. Pharmacological inhibitors of RAC1 and CDC42 most efficiently attenuated DOX-induced DNA damage in all cell types examined in vitro. Consistently, immunohistochemical analyses revealed that the RAC1 inhibitor NSC23766 and the pan-RHO GTPase inhibitor lovastatin reduced the level of DOX-induced residual DNA damage in both cardiomyocytes and non-cardiomyocytes in vivo. Overall, we conclude that endothelial cells, fibroblasts and cardiomyocytes contribute to the pathophysiology of DOX-induced cardiotoxicity, with RAC1- and CDC42-regulated signaling pathways being especially relevant for DOX-stimulated DSB formation and DNA damage response (DDR) activation. Hence, we suggest dual targeting of RAC1/CDC42-dependent mechanisms in multiple cardiac cell types to mitigate DNA damage-dependent cardiac injury evoked by DOX-based anticancer therapy.

DNA Double-Strand Break Response and Repair Gene Polymorphisms May Influence Therapy Results and Prognosis in Head and Neck Cancer Patients.

Radiotherapy and cisplatin-based chemotherapy belong to the main treatment modalities for head and neck squamous cell carcinoma (HNSCC) and induce cancer cell death by generating DNA damage, including the most severe double-strand breaks (DSBs). Alterations in DSB response and repair genes may affect individual DNA repair capacity and treatment sensitivity, contributing to the therapy resistance and poor prognosis often observed in HNSCC. In this study, we investigated the association of a panel of single-nucleotide polymorphisms (SNPs) in 20 DSB signaling and repair genes with therapy results and prognosis in 505 HNSCC patients treated non-surgically with DNA damage-inducing therapies. In the multivariate analysis, there were a total of 14 variants associated with overall, locoregional recurrence-free or metastasis-free survival. Moreover, we identified 10 of these SNPs as independent predictors of therapy failure and unfavorable prognosis in the whole group or in two treatment subgroups. These were MRE11 rs2155209, XRCC5 rs828907, RAD51 rs1801321, rs12593359, LIG4 rs1805388, CHEK1 rs558351, TP53 rs1042522, ATM rs1801516, XRCC6 rs2267437 and NBN rs2735383. Only CHEK1 rs558351 remained statistically significant after correcting for multiple testing. These results suggest that specific germline variants related to DSB response and repair may be potential genetic modifiers of therapy effects and disease progression in HNSCC treated with radiotherapy and cisplatin-based chemoradiation.

Pre-rRNA Facilitates TopBP1-Mediated DNA Double-Strand Break Response.

In response to genotoxic stress-induced DNA damage, TopBP1 mediates ATR activation for signaling transduction and DNA damage repair. However, the detailed molecular mechanism remains elusive. Here, using unbiased protein affinity purification and RNA sequencing, it is found that TopBP1 is associated with pre-ribosomal RNA (pre-rRNA). Pre-rRNA co-localized with TopBP1 at DNA double-strand breaks (DSBs). Similar to pre-rRNA, ribosomal proteins also colocalize with TopBP1 at DSBs. The recruitment of TopBP1 to DSBs is suppressed when cells are transiently treated with RNA polymerase I inhibitor (Pol I-i) to suppress pre-rRNA biogenesis but not protein translation. Moreover, the BRCT4-5 of TopBP1 recognizes pre-rRNA and forms liquid-liquid phase separation (LLPS) with pre-rRNA, which may be the molecular basis of DSB-induced foci of TopBP1. Finally, Pol I-i treatment impairs TopBP1-associated cell cycle checkpoint activation and homologous recombination repair. Collectively, this study reveals that pre-rRNA plays a key role in the TopBP1-dependent DNA damage response.

Multiparametric analysis of etoposide exposed mesenchymal stem cells and Fanconi anemia cells: implications in development of secondary myeloid malignancy.

Secondary acute myeloid leukemia (sAML) may develop following a prior therapy or may evolve from an antecedent hematological disorder such as Fanconi Anemia (FA). Pathophysiology of leukemic evolution is not clear. Etoposide (Eto) is a chemotherapeutic agent implicated in development of sAML. FA is an inherited bone marrow (BM) failure disease characterized by genomic instability and xenobiotic susceptibility. Here, we hypothesized that alterations in the BM niche may play a critical/driver role in development of sAML in both conditions. Expression of selected genes involved in xenobiotic metabolism, DNA double-strand break response, endoplasmic reticulum (ER) stress, heat shock response and cell cycle regulation were determined in BM mesenchymal stem cells (MSCs) of healthy controls and FA patients at steady state and upon exposure to Eto at different concentrations and in recurrent doses. Expression of CYPA1, p53, CCNB1, Dicer1, CXCL12, FLT3L and TGF-Beta genes were significantly downregulated in FA-MSCs compared with healthy controls. Eto exposure induced significant alterations in healthy BM-MSCs with increased expression of CYP1A1, GAD34, ATF4, NUPR1, CXCL12, KLF4, CCNB1 and nuclear localization of Dicer1. Interestingly, FA-MSCs did not show significant alterations in these genes upon Eto exposure. As opposed to healthy MSCs DICER1 gene expression and intracellular localization was not altered on FA BM-MSCs after Eto treatment. These results showed that Eto is a highly potent molecule and has pleiotropic effects on BM-MSCs, FA cells show altered expression profile compared to healthy controls and Eto exposure on FA cells shows differential profile than healthy controls.

Selected Articles from This Issue

Highlights| March 01 2023 Selected Articles from This Issue Author & Article Information Online ISSN: 1557-3125 Print ISSN: 1541-7786 ©2023 American Association for Cancer Research2023American Association for Cancer Research Mol Cancer Res (2023) 21 (3): 187. https://doi.org/10.1158/1541-7786.MCR-21-3-HI Related Content A commentary has been published: Tumor-Suppressive and Immune-Stimulating Roles of Cholesterol 25-hydroxylase in Pancreatic Cancer Cells A commentary has been published: A Novel Tumor-Promoting Role for Nuclear Factor IX in Glioblastoma Is Mediated through Transcriptional Activation of GINS1 A commentary has been published: ATM Regulation of the Cohesin Complex Is Required for Repression of DNA Replication and Transcription in the Vicinity of DNA Double-Strand Breaks View more A commentary has been published: MUC1-C Dictates PBRM1-Mediated Chronic Induction of Interferon Signaling, DNA Damage Resistance, and Immunosuppression in Triple-Negative Breast Cancer View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record March 1 2023 Citation Selected Articles from This Issue. Mol Cancer Res 1 March 2023; 21 (3): 187. https://doi.org/10.1158/1541-7786.MCR-21-3-HI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Ataxia telangiectasia mutated (ATM) orchestrates DNA double-strand break (DSB) responses by phosphorylating proteins that regulate DNA damage repair (DDR). ATM mutations potentiate oncogenic transformation and characterizing the ATM interactome may unveil novel mechanisms underlying DDR and carcinogenesis. Using proteomic screens, Bass and colleagues discovered novel interactions between ATM and cohesin complex constituents, including PDS5A. The authors confirmed that ATM phosphorylates PDS5A using a pharmacologic ATM inhibitor and Western blotting. Moreover, they found that phosphomutant PDS5A transfection increases irradiation-induced DNA damage, suggesting ATM-mediated PDS5A phosphorylation promotes DDR. Using a reporter cell line in which DSB and DDR-induced transcription repression can be tracked using red and yellow fluorescence, respectively, the authors showed that ATM-mediated PDS5A phosphorylation facilitates transcription repression in response to DSBs. The authors also developed a novel S-phase checkpoint assay capable of measuring 5-ethynyl-2’-deoxyuridine (EdU) incorporation in the vicinity of fluorescent DSB radii and showed that PDS5A phosphorylation slows DNA... You do not currently have access to this content.

Preliminary Study on 53BP1-Mediated DNA Double-Strand Break Response in Spermatogonial Stem Cells.

53BP1 mediates DNA repair process in somatic cells; however, the function of 53BP1 in germline stem cells still remains unclear. In the present study, animals and cells DNA damage repair (DDR) model was established by irradiation and HU treatment; immunofluorescence staining and laser confocal microscopy were used to detect the expression of 53BP1, p-CHK2, and p-P53 in the DDR process of mSSCs. 53BP1 knockdown expression mSSCs cell line conducted by Trp53bp1-shRNA was established and EdU staining was adopted to analyze cell cycle and cell proliferation. Moreover, NHEJ reporter vector was applied to detect the repair efficacy after Trp53bp1 knocked-down (KD) expression. Results showed that 53BP1 could form foci signals in mSSCs during DDR process both in vivo and in vitro, which was independent of γH2AX. 53BP1 downstream protein, p-P53, and p-CHK2 were involved and dynamically expressed in DDR response. Knocking down of Trp53bp1 expression in mSSCs could not dramatically inhibit cell proliferation, but may increase cell sensitivity to HU. The NHEJ repair efficacy was sharply decreased in Trp53bp-KD SSCs via flow cytometry analysis. We revealed the specific mechanism of 53BP1 in SSCs DDR process, which is expected to provide a new theoretical basis and insights for the diagnosis and treatment of male infertility.

Deubiquitinase USP2 stabilizes the MRE11–RAD50–NBS1 complex at DNA double-strand break sites by counteracting the ubiquitination of NBS1

The MRE11-RAD50-NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown. Here, we showed that USP2 counteracts this ubiquitination and stabilizes the MRN complex during the DSB response. By screening deubiquitinases that increase the stability of the MRN complex in RECQL4-deficient cells, USP2 was identified as a new deubiquitinase that acts at DSB sites to counteract NBS1 ubiquitination. We determined that USP2 is recruited to DSB sites in a manner dependent on ATM, a major checkpoint kinase against DSBs, and stably interacts with NBS1 and RECQL4 in immunoprecipitation experiments. Phosphorylation of two critical residues in the N terminus of USP2 by ATM is required for its recruitment to DSBs and its interaction with RECQL4. While inactivation of USP2 alone does not substantially influence the DSB response, we found that inactivation of USP2 and USP28, another deubiquitinase influencing NBS1 ubiquitination, results in premature disassembly of the MRN complex from DSB sites as well as defects in ATM activation and homologous recombination repair abilities. These results suggest that deubiquitinases counteracting NBS1 ubiquitination are essential for the stable maintenance of the MRN complex and proper cellular response to DSBs.

Unveiling the Molecular Mechanisms Driving the Capsaicin-Induced Immunomodulatory Effects on PD-L1 Expression in Bladder and Renal Cancer Cell Lines.

Simple SummaryOver time, capsaicin (CPS) has been considered both a potential anti-cancer and pro-cancer molecule. Hence, the diversity of CPS functioning has already been established. Now, exploration of its application with immunotherapies might open up a new avenue in cancer therapy. Herein, the application of CPS as an immunoadjuvant to overcome the tumor’s immune-escaping mechanisms or to increase immune checkpoint therapy has been approached. In bladder cancer, the interaction of CPS with its receptor TRPV1 increases PD-L1 expression, promoting a tumorigenic effect and also providing a target for anti-PD-1/PD-L1 immunotherapy. On the contrary, in renal cell carcinoma, CPS downregulates PD-L1 expression in a TRPV1-independent manner, suggesting a potential application of CPS as an immune-adjuvant in this type of cancer.The blockade of the PD-L1/PD-1 immune checkpoint has promising efficacy in cancer treatment. However, few patients with bladder cancer (BC) or renal cell carcinoma (RCC) respond to this approach. Thus, it is important to implement a strategy to stimulate the immune anti-tumor response. In this scenario, our study evaluated the effects of a low capsaicin (CPS) dose in BC and RCC cell lines. Western blot, qRT-PCR and confocal microscopy were used to assess PD-L1 mRNA and protein expression. Alterations to the cellular oxidative status and changes to the antioxidant NME4 levels, mRNA modulation of cytokines, growth factors, transcriptional factors and oncogene, and the activation of Stat1/Stat3 pathways were examined using Western blot, cytofluorimetry and qRT-PCR profiling assays. In BC, CPS triggers an altered stress oxidative-mediated DNA double-strand break response and increases the PD-L1 expression. On the contrary, in RCC, CPS, by stimulating an efficient DNA damage repair response, thus triggering protein carbonylation, reduces the PD-L1 expression. Overall, our results show that CPS mediates a multi-faceted approach. In modulating PD-L1 expression, there is a rationale for CPS exploitation as a stimulus that increases BC cells’ response to immunotherapy or as an immune adjuvant to improve the efficacy of the conventional therapy in RCC patients.

Conserved function of Drosophila Fancd2 monoubiquitination in response to double-strand DNA breaks.

Fanconi anemia genes play key roles in metazoan DNA damage responses, and human FA mutations cause numerous disease phenotypes. In human cells, activating monoubiquitination of the Fanconi anemia protein Fancd2 occurs following diverse DNA damage stimuli. Monoubiquitinated Fancd2 forms nuclear foci to recruit additional repair factors. Fancd2 animal models to date have focused on molecular nulls or whole gene knockdown, leaving the specific in vivo role of monoubiquitination unclear. Using a point mutant in a conserved residue, we recently linked Drosophila Fancd2 monoubiquitination to a mitosis-specific DNA double-strand break response. In this context, we used CRISPR/Cas9 to generate the first animal model of an endogenous mutation in the conserved monoubiquitination site (fancd2K595R). Here, we expand upon our characterization of fancd2K595R. We also introduce and characterize additional Drosophila tools to study fancd2, including new mutant alleles and GFP-tagged rescue transgenes. Using these new reagents, we show the impact of Drosophila Fancd2 on organismal and cell viability, as well as on repair protein localization, in the presence or absence of double-strand breaks. These findings expand our understanding of Fanconi anemia gene function in vivo and provide useful reagents for DNA repair research.

MRNIP condensates promote DNA double-strand break sensing and end resection

The rapid recognition of DNA double-strand breaks (DSBs) by the MRE11/RAD50/NBS1 (MRN) complex is critical for the initiation of DNA damage response and DSB end resection. Here, we show that MRN complex interacting protein (MRNIP) forms liquid-like condensates to promote homologous recombination-mediated DSB repair. The intrinsically disordered region is essential for MRNIP condensate formation. Mechanically, the MRN complex is compartmentalized and concentrated into MRNIP condensates in the nucleus. After DSB formation, MRNIP condensates move to the damaged DNA rapidly to accelerate the binding of DSB by the concentrated MRN complex, therefore inducing the autophosphorylation of ATM and subsequent activation of DNA damage response signaling. Meanwhile, MRNIP condensates-enhanced MRN complex loading further promotes DSB end resection. In addition, data from xenograft models and clinical samples confirm a correlation between MRNIP and radioresistance. Together, these results reveal an important role of MRNIP phase separation in DSB response and the MRN complex-mediated DSB end resection.

The roles of HMGB1-produced DNA gaps in DNA protection and aging biomarker reversal.

The endogenous DNA damage triggering an aging progression in the elderly is prevented in the youth, probably by naturally occurring DNA gaps. Decreased DNA gaps are found during chronological aging in yeast. So we named the gaps “Youth‐DNA‐GAPs.” The gaps are hidden by histone deacetylation to prevent DNA break response and were also reduced in cells lacking either the high‐mobility group box (HMGB) or the NAD‐dependent histone deacetylase, SIR2. A reduction in DNA gaps results in shearing DNA strands and decreasing cell viability. Here, we show the roles of DNA gaps in genomic stability and aging prevention in mammals. The number of Youth‐DNA‐GAPs were low in senescent cells, two aging rat models, and the elderly. Box A domain of HMGB1 acts as molecular scissors in producing DNA gaps. Increased gaps consolidated DNA durability, leading to DNA protection and improved aging features in senescent cells and two aging rat models similar to those of young organisms. Like the naturally occurring Youth‐DNA‐GAPs, Box A‐produced DNA gaps avoided DNA double‐strand break response by histone deacetylation and SIRT1, a Sir2 homolog. In conclusion, Youth‐DNA‐GAPs are a biomarker determining the DNA aging stage (young/old). Box A‐produced DNA gaps ultimately reverse aging features. Therefore, DNA gap formation is a potential strategy to monitor and treat aging‐associated diseases.

DNA Double-Strand Break Repairs and Their Application in Plant DNA Integration.

Double-strand breaks (DSBs) are considered to be one of the most harmful and mutagenic forms of DNA damage. They are highly toxic if unrepaired, and can cause genome rearrangements and even cell death. Cells employ two major pathways to repair DSBs: homologous recombination (HR) and non-homologous end-joining (NHEJ). In plants, most applications of genome modification techniques depend on the development of DSB repair pathways, such as Agrobacterium-mediated transformation (AMT) and gene targeting (GT). In this paper, we review the achieved knowledge and recent advances on the DNA DSB response and its main repair pathways; discuss how these pathways affect Agrobacterium-mediated T-DNA integration and gene targeting in plants; and describe promising strategies for producing DSBs artificially, at definite sites in the genome.

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.

Nemo-Dependent, ATM-Mediated Signals from RAG DNA Breaks at Igk Feedback Inhibit V κ Recombination to Enforce Igκ Allelic Exclusion.

Monoallelic AgR gene expression underlies specific adaptive immune responses. AgR allelic exclusion is achieved by sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. The ATM kinase, a regulator of the DNA double-strand break (DSB) response, helps enforce allelic exclusion through undetermined mechanisms. ATM promotes repair of RAG1/RAG2 (RAG) endonuclease-induced DSBs and transduces signals from RAG DSBs during Igk gene rearrangement on one allele to transiently inhibit RAG1 protein expression, Igk accessibility, and RAG cleavage of the other allele. Yet, the relative contributions of ATM functions in DSB repair versus signaling to enforce AgR allelic exclusion remain undetermined. In this study, we demonstrate that inactivation in mouse pre-B cells of the NF-κB essential modulator (Nemo) protein, an effector of ATM signaling, diminishes RAG DSB-triggered repression of Rag1/Rag2 transcription and Igk accessibility but does not result in aberrant repair of RAG DSBs like ATM inactivation. We show that Nemo deficiency increases simultaneous biallelic Igk cleavage in pre-B cells and raises the frequency of B cells expressing Igκ proteins from both alleles. In contrast, the incidence of biallelic Igκ expression is not elevated by inactivation of the SpiC transcriptional repressor, which is induced by RAG DSBs in an ATM-dependent manner and suppresses Igk accessibility. Thus, we conclude that Nemo-dependent, ATM-mediated DNA damage signals enforce Igκ allelic exclusion by orchestrating transient repression of RAG expression and feedback inhibition of additional Igk rearrangements in response to RAG cleavage on one Igk allele.

Pre-ribosomal RNA reorganizes DNA damage repair factors in nucleus during meiotic prophase and DNA damage response.

In response to DNA double-strand breaks (DSBs), DNA damage repair factors are recruited to DNA lesions and form nuclear foci. However, the underlying molecular mechanism remains largely elusive. Here, by analyzing the localization of DSB repair factors in the XY body and DSB foci, we demonstrate that pre-ribosomal RNA (pre-rRNA) mediates the recruitment of DSB repair factors around DNA lesions. Pre-rRNA exists in the XY body, a DSB repair hub, during meiotic prophase, and colocalizes with DSB repair factors, such as MDC1, BRCA1 and TopBP1. Moreover, pre-rRNA-associated proteins and RNAs, such as ribosomal protein subunits, RNase MRP and snoRNAs, also localize in the XY body. Similar to those in the XY body, pre-rRNA and ribosomal proteins also localize at DSB foci and associate with DSB repair factors. RNA polymerase I inhibitor treatment that transiently suppresses transcription of rDNA but does not affect global protein translation abolishes foci formation of DSB repair factors as well as DSB repair. The FHA domain and PST repeats of MDC1 recognize pre-rRNA and mediate phase separation of DSB repair factors, which may be the molecular basis for the foci formation of DSB repair factors during DSB response.

Role of the Histone Acetyl Transferase MOF and the Histone Deacetylase Sirtuins in Regulation of H4K16ac During DNA Damage Repair and Metabolic Programming: Implications in Cancer and Aging.

The accurate repair of genomic damage mediated by ionizing radiation (IR), chemo- or radiomimetic drugs, or other exogenous agents, is necessary for maintenance of genome integrity, preservation of cellular viability and prevention of oncogenic transformation. Eukaryotes have conserved mechanisms designed to perceive and repair the damaged DNA quite efficiently. Among the different types of DNA damage, double strand breaks (DSB) are the most detrimental. The cellular DNA DSB response is a hierarchical signaling network that integrates damage sensing and repair with chromatin structural changes that involve a range of pre-existing and induced covalent modifications. Recent studies have revealed that pre-existing histone modifications are important contributors within this signaling/repair network. This chapter discusses the role of a critical histone acetyl transferase (HAT) known as MOF (males absent on the first) and the histone deacetylases (HDACs) Sirtuins on histone H4K16 acetylation (H4K16ac)and DNA damage repair. We also discuss the role of this important histone modification in light of metabolic rewiring and its role in regulating human pathophysiologic states.

Stable maintenance of the Mre11-Rad50-Nbs1 complex is sufficient to restore the DNA double-strand break response in cells lacking RecQL4 helicase activity

The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund–Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.

Identification of a Compound Overcoming Drug Resistance of Myeloma Cells at Hypoxic Condition

IntroductionHypoxia plays a crucial role in the development of drug resistance in various cancer cells. Therefore, many attempts targeting hypoxia are underway to overcome the drug resistance mediated by hypoxia. This strategy is useful for multiple myeloma (MM) cells, as MM cells reside within the bone marrow, where oxygen concentrations are quite low. To elucidate influence of oxygen concentration to drug sensitivity of MM cells, we evaluated sensitivity of MM cells to lenalidomide and melphalan under hypoxia (oxygen concentration: 1%). We found that significant elevation of LD50 for MM cell lines, KMS12PE and U266, were observed (Fig. 1), indicating drug resistances to these reagents are indeed induced under hypoxia.Materials and MethodsMM cell lines, KMS12PE and U266, were utilized. Natural compound library was obtained from Institute of Natural Medicine, Toyama University (Toyama, Japan). Cell viability was analyzed either by MTT assay or trypan blue dye eclusion analysis. DNA double strand break response was analyzed by western blot detetcting gamma-H2AX. Melphalan was utilized as an conventional anti-MM reagent causing DNA double strand break.Results and DiscussionWe screened natural compound library to identify compounds exerting cytotoxicity in MM cells under hypoxic condition. Bufalin, a component of a chinese medicine (Chan Su), exhibited marked cytotoxicity to MM cells under both hypoxic and normoxic conditions at nano molar levels (compound#5 in Fig. 2). Importantly, no significant toxicity by bufalin was observed for PBMC obtained from normal donors.At normoxic condition, bufalin induced significant DNA double strand break (DSB) response, ROS induction and caspase activation within 24 hours which is a more rapid response compared with melphalan. Interestingly, the bufalin-induced DSB response was not impaired by low oxygen concentrations while DSB response by melphalan was reduced (Fig. 3). Moreover, treatment with bufalin abolished HIF-1α expression under hypoxia (Fig. 4), suggesting bufalin exerts cytotoxicity under hypoxia by regulating HIF-1α.These results suggest that bufalin induces apoptosis to MM cells through DSB under hypoxic condition by inhibiting HIF-1α, suggesting bufalin could be useful for eradication of drug resistant MM cells in the hypoxic microenvironment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

New Faces of old Friends: Emerging new Roles of RNA-Binding Proteins in the DNA Double-Strand Break Response.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. To protect genomic stability and ensure cell homeostasis, cells mount a complex signaling-based response that not only coordinates the repair of the broken DNA strand but also activates cell cycle checkpoints and, if necessary, induces cell death. The last decade has seen a flurry of studies that have identified RNA-binding proteins (RBPs) as novel regulators of the DSB response. While many of these RBPs have well-characterized roles in gene expression, it is becoming increasingly clear that they also have non-canonical functions in the DSB response that go well beyond transcription, splicing and mRNA processing. Here, we review the current understanding of how RBPs are integrated into the cellular response to DSBs and describe how these proteins directly participate in signal transduction, amplification and repair at damaged chromatin. In addition, we discuss the implications of an RBP-mediated DSB response for genome instability and age-associated diseases such as cancer and neurodegeneration.

Histone sumoylation and chromatin dynamics.

Chromatin structure and gene expression are dynamically controlled by post-translational modifications (PTMs) on histone proteins, including ubiquitylation, methylation, acetylation and small ubiquitin-like modifier (SUMO) conjugation. It was initially thought that histone sumoylation exclusively suppressed gene transcription, but recent advances in proteomics and genomics have uncovered its diverse functions in cotranscriptional processes, including chromatin remodeling, transcript elongation, and blocking cryptic initiation. Histone sumoylation is integral to complex signaling codes that prime additional histone PTMs as well as modifications of the RNA polymerase II carboxy-terminal domain (RNAPII-CTD) during transcription. In addition, sumoylation of histone variants is critical for the DNA double-strand break (DSB) response and for chromosome segregation during mitosis. This review describes recent findings on histone sumoylation and its coordination with other histone and RNAPII-CTD modifications in the regulation of chromatin dynamics.