Levels Of DNA Double-strand Breaks Research Articles

Low levels of endogenous or X-ray-induced DNA double-strand breaks activate apoptosis in adult neural stem cells

ABSTRACT The embryonic neural stem cell compartment is characterised by rapid proliferation from embryonic day (E)11 to E16.5, high endogenous DNA double-strand break (DSB) formation and sensitive activation of apoptosis. Here, we ask whether DSBs arise in the adult neural stem cell compartments, the sub-ventricular zone (SVZ) of the lateral ventricles and the sub-granular zone (SGZ) of the hippocampal dentate gyrus, and whether they activate apoptosis. We used mice with a hypomorphic mutation in DNA ligase IV ( Lig4 Y288C ), ataxia telangiectasia mutated ( Atm − / − ) and double mutant Atm − / − / Lig4 Y288C mice. We demonstrate that, although DSBs do not arise at a high frequency in adult neural stem cells, the low numbers of DSBs that persist endogenously in Lig 4 Y288C mice or that are induced by low radiation doses can activate apoptosis. A temporal analysis shows that DSB levels in Lig4 Y288C mice diminish gradually from the embryo to a steady state level in adult mice. The neonatal SVZ compartment of Lig4 Y288C mice harbours diminished DSBs compared to its differentiated counterpart, suggesting a process selecting against unfit stem cells. Finally, we reveal high endogenous apoptosis in the developing SVZ of wild-type newborn mice.

A surge of late-occurring meiotic double-strand breaks rescues synapsis abnormalities in spermatocytes of mice with hypomorphic expression of SPO11.

Meiosis is the biological process that, after a cycle of DNA replication, halves the cellular chromosome complement, leading to the formation of haploid gametes. Haploidization is achieved via two successive rounds of chromosome segregation, meiosis I and II. In mammals, during prophase of meiosis I, homologous chromosomes align and synapse through a recombination-mediated mechanism initiated by the introduction of DNA double-strand breaks (DSBs) by the SPO11 protein. In male mice, if SPO11 expression and DSB number are reduced below heterozygosity levels, chromosome synapsis is delayed, chromosome tangles form at pachynema, and defective cells are eliminated by apoptosis at epithelial stage IV at a spermatogenesis-specific endpoint. Whether DSB levels produced in Spo11+/− spermatocytes represent, or approximate, the threshold level required to guarantee successful homologous chromosome pairing is unknown. Using a mouse model that expresses Spo11 from a bacterial artificial chromosome, within a Spo11−/− background, we demonstrate that when SPO11 expression is reduced and DSBs at zygonema are decreased (approximately 40 % below wild-type level), meiotic chromosome pairing is normal. Conversely, DMC1 foci number is increased at pachynema, suggesting that under these experimental conditions, DSBs are likely made with delayed kinetics at zygonema. In addition, we provide evidences that when zygotene-like cells receive enough DSBs before chromosome tangles develop, chromosome synapsis can be completed in most cells, preventing their apoptotic elimination.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-015-0544-7) contains supplementary material, which is available to authorized users.

Abstract 4739: Genetic predisposition to DNA double strand break repair defect defines a new class of familial colon cancer

Abstract Colorectal cancer (CRC) is the second most common fatal cancer in the U.S., with ∼ 30% cases familial (FCRC). Genetic predispositions have only been defined for 25% of FCRC (including Lynch syndrome, Familial Adenomatous Polyposis (FAP), and MutYH polyposis). For the genetically undefined group (uFCRC), understanding of disease pathogenesis is limited, and the absence of defined defects in relevant pathways that could support molecular diagnosis results in under- and over-screening. We hypothesized that uFCRC would arise distinctly from moderately penetrant germline defects in repair of DNA double-strand breaks (DSBs). Such defects in DSB repair would lead to a moderate level of constitutional genomic instability (CGI) that could be assayed from patient peripheral blood lymphocytes (PBLs). We further hypothesized that such DNA lesions would often be heterozygous, associated with haploinsufficiency or dominant-negative effects, and would impair protein interactions important for DNA repair. We combined exome sequencing with biological assays to screen a rich resource of uFCRC clinical samples from the Fox Chase Cancer Center Risk Assessment Program. Exome sequencing of PBLs from patients (N = 25) identified a mean of 1.4 rare variants/patient that were 1) strongly predicted to damage protein function using multiple functional predictors (SIFT, Polyphen-2, Provean, and MutationAssessor), 2) involved in pathways that suppress DSBs, and 3) have not been previously implicated in CRC. Further, many of the variant genes were commonly mutated in CRCs and other cancers in TCGA database. Comparison of PBLs from patients and age- and sex- matched controls revealed increased DSB levels (basal and with DNA damaging agents) in 18/25 patients vs. 1/25 normal controls (P = 0.001, AUC = 0.85). Next, knockdown of variant genes in HCT116 CRC cells increased DSB formation with or without damaging agents. As a proof-of-concept, we studied one patient in detail who had candidate disease-causing variants in 2 DNA repair genes, ERCC6 and WRN. Both variants were dysfunctional in biochemical tests. We established patient-derived EBV-lines and observed, at baseline and with several DNA damaging agents, elevated levels of γH2AX foci and larger nuclear comets, indicative of DSBs. This phenotype could be rescued by expression of wild type WRN or ERCC6 and conversely induced by knockdown in CRC cells. Further, the patient derived lines exhibited reduced expression of WRN and ERCC6, implicating haploinsufficiency of these genes in the phenotype observed. Our data support the hypothesis that defects in DSB repair genes cause moderate-level CGI that commonly presents as uFCRC. Our studies identify novel genetic subsets of FCRC, and could potentially predict familial risk and improve screening and diagnosis. Citation Format: sanjeevani arora, Hong Yan, Iltaeg Cho, Hua-Ying Fan, Biao Luo, xiaowu gai, dale bodian, joe vockley, yan zhou, elizabeth handorf, mark andrake, emmanuelle nicolas, Ilya Serebriiskii, tim yen, michael hall, greg enders, erica golemis. Genetic predisposition to DNA double strand break repair defect defines a new class of familial colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4739. doi:10.1158/1538-7445.AM2015-4739

Streptococcus pneumoniae secretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H2O2, plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H2O2, greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H2O2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniae-induced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia.

PTH-336 Effects of lower doses of dca on radiosensitisation of colorectal cancer cells in vitro

Introduction Neoadjuvant radiotherapy confers a well-established benefit in locoregional recurrence for patients with locally invasive rectal cancer. However, only around 50% experience significant downstaging of their tumour. Radiation-associated toxicity renders dose escalation unachievable. Dichloroacetate (DCA) is a generic drug shown to modify tumour metabolism reversing the Warburg effect and inducing apoptosis and may potentially enhance the effect of radiation. This study aimed to investigate the effects of DCA at lower doses in combination with radiotherapy on the survival of CRC cell lines, and any possible synergistic DNA damage. Method CRC cells (LoVo) and control cells (HEK 293) treated with DCA (2,5,10 mM) and irradiation (0,4,8 Gy). Influences of the combination treatment on cell survival were analysed using MTT assays and clonogenic assays and the induction of DNA damage by quantifying gamma-H2AX foci. Statistical analyses were performed using GraphPad, differences between DCA-treated and vehicle control groups were assessed using the Mann-Whitney U test. Results DCA (5 mM) significantly reduced the survival of CRC cells in combination with 4 Gy and 8 Gy (P = 0.0244, 0.0078, respectively). DCA (10 mM) significantly reduced survival of CRC cells in in combination with 4 Gy and 8 Gy (P DCA (5 mM) significantly increased the mean number of DNA double-strand breaks (DSB) per CRC cell in combination with 4 Gy and 8 Gy (P = 0.0041, 0.023, respectively). In unirradiated CRC cells, all the DCA doses (2, 5, 10 mM) significantly increased the mean number of DSB’s per CRC cell (P Conclusion Lower doses of DCA may have an additive killing effect in combination with radiation in CRC cells. A similar picture of sensitivity was seen in normal cell line demonstrating that DCA is not completely harmless. Increasing doses of DCA associated with increased levels of DSB’s in CRC cells and it may be a potential mechanism behind DCA. Further research is warranted exploring the mechanism behind the effects of DCA and as a potential adjunct in the management of rectal cancers. Disclosure of interest None Declared.

Persistent DNA Damage in Spermatogonial Stem Cells After Fractionated Low-Dose Irradiation of Testicular Tissue

Testicular spermatogenesis is extremely sensitive to radiation-induced damage, and even low scattered doses to testis from radiation therapy may pose reproductive risks with potential treatment-related infertility. Radiation-induced DNA double-strand breaks (DSBs) represent the greatest threat to the genomic integrity of spermatogonial stem cells (SSCs), which are essential to maintain spermatogenesis and prevent reproduction failure. During daily low-dose radiation with 100mGy or 10mGy, radiation-induced DSBs were monitored in mouse testis by quantifying 53 binding protein 1 (53BP-1) foci in SSCs within their stem cell niche. The accumulation of DSBs was correlated with proliferation, differentiation, and apoptosis of testicular germ cell populations. Even very low doses of ionizing radiation arrested spermatogenesis, primarily by inducing apoptosis in spermatogonia. Eventual recovery of spermatogenesis depended on the survival of SSCs and their functional ability to proliferate and differentiate to provide adequate numbers of differentiating spermatogonia. Importantly, apoptosis-resistant SSCs resulted in increased 53BP-1 foci levels during, and even several months after, fractionated low-dose radiation, suggesting that surviving SSCs have accumulated an increased load of DNA damage. SSCs revealed elevated levels of DSBs for weeks after radiation, and if these DSBs persist through differentiation to spermatozoa, this may have severe consequences for the genomic integrity of the fertilizing sperm.

Assessment of modulated cytostatic drug resistance by automated γH2AX analysis.

The efficacy of many chemotherapeutic agents relies on the preferential destruction of rapidly dividing cancer cells by inducing various kinds of DNA damage. The most deleterious type of DNA lesions are DNA double-strand breaks (DSB), which can be detected by immunofluorescence staining of phosphorylated histone protein H2AX (γH2AX). Furthermore, γH2AX has been suggested as clinical pharmacodynamic biomarker in chemotherapeutic cancer treatment. A great challenge in treating neoplastic diseases is the varying response behavior among cancer patients. Thus, intrinsic or drug-induced overexpression of efflux pumps often leads to multiple drug resistance (MDR) and treatment failure. In particular, inter-individual differences in expression levels of efflux pumps, such as the permeability glycoprotein (P-gp), were shown to correlate with cancer progression. Several efficient cytostatic drugs, including the DSB-inducing agent etoposide (ETP) are known P-gp substrates. In this respect, modulation of MDR by P-gp inhibitors, like the immunosuppressives cyclosporine A (CsA) and rapamycin (Rapa) have been described. Here, we investigated the application of γH2AX focus assay to monitor the impact of CsA and Rapa on ETP-induced cytotoxicity in human peripheral blood mononuclear cells. Evaluation of γH2AX foci was performed by the automated fluorescence microscopy and interpretation system AKLIDES. Compared to ETP treatment alone, our results revealed a significant rise in γH2AX focus number and percentage of DSB-positive cells after cells have been treated with ETP in the presence of either CsA or Rapa. In contrast, DSB levels of cells incubated with CsA or Rapa alone were comparable to focus number of untreated cells. Our results successfully demonstrated how automated γH2AX analysis can be used as fast and reliable approach to monitor drug resistance and the impact of MDR modulators during treatment with DSB-inducing cytostatics..

Ionizing radiation-induced cell death is partly caused by increase of mitochondrial reactive oxygen species in normal human fibroblast cells.

Radiation-induced cell death is thought to be caused by nuclear DNA damage that cannot be repaired. However, in this study we found that a delayed increase of mitochondrial reactive oxygen species (ROS) is responsible for some of the radiation-induced cell death in normal human fibroblast cells. We have previously reported that there is a delayed increase of mitochondrial (·)O2(-), measured using MitoSOX™ Red reagent, due to gamma irradiation. This is dependent on Drp1 localization to mitochondria. Here, we show that knockdown of Drp1 expression reduces the level of DNA double-strand breaks (DSBs) remaining 3 days after 6 Gy irradiation. Furthermore, cells with knockdown of Drp1 expression are more resistant to gamma radiation. We then tested whether the delayed increase of ROS causes DNA damage. The antioxidant, 2-glucopyranoside ascorbic acid (AA-2G), was applied before or after irradiation to inhibit ROS production during irradiation or to inhibit delayed ROS production from mitochondria. Interestingly, 1 h after exposure, the AA-2G treatment reduced the level of DSBs remaining 3 days after 6 Gy irradiation. In addition, irradiated AA-2G-treated cells were more resistant to radiation than the untreated cells. These results indicate that delayed mitochondrial ROS production may cause some of the cell death after irradiation.

P53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage.

The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.

NAD+ treatment can prevent rotenone-induced increases in DNA damage, Bax levels and nuclear translocation of apoptosis-inducing factor in differentiated PC12 cells.

Nicotinamide adenine dinucleotide (NAD(+)) plays critical roles in energy metabolism, mitochondrial functions, calcium homeostasis and immunological functions. Our previous studies have found that NAD(+) administration can profoundly decrease ischemic brain injury and traumatic brain injury. Our recent study has also provided first direct evidence indicating that NAD(+) treatment can decrease cellular apoptosis, while the mechanisms underlying this protective effect remain unclear. In our current study, we determined the effects of NAD(+) treatment on several major factors in apoptosis and necrosis, including levels of Bax and nuclear translocation of apoptosis-inducing factor (AIF), as well as levels of DNA double-strand breaks (DSBs) and intracellular ATP in rotenone-treated differentiated PC12 cells. We found that NAD(+) treatment can markedly attenuate the rotenone-induced increases in the levels of Bax and nuclear translocation of AIF in the cells. We further found that NAD(+) treatment can significantly attenuate the rotenone-induced increase in the levels of DSBs and decrease in the intracellular ATP levels. Collectively, our study has suggested mechanisms underlying the preventive effects of NAD(+) on apoptosis, which has highlighted the therapeutic potential of NAD(+) for decreasing apoptotic changes in multiple major diseases.

The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity.

In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O(6)-methylguanine (O(6)MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O(6)-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O(6)-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O(6)-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O(6)MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O(6)MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.

Low levels of endogenous or X-ray-induced DNA double-strand breaks activate apoptosis in adult neural stem cells.

ABSTRACTThe embryonic neural stem cell compartment is characterised by rapid proliferation from embryonic day (E)11 to E16.5, high endogenous DNA double-strand break (DSB) formation and sensitive activation of apoptosis. Here, we ask whether DSBs arise in the adult neural stem cell compartments, the sub-ventricular zone (SVZ) of the lateral ventricles and the sub-granular zone (SGZ) of the hippocampal dentate gyrus, and whether they activate apoptosis. We used mice with a hypomorphic mutation in DNA ligase IV (Lig4Y288C), ataxia telangiectasia mutated (Atm−/−) and double mutant Atm−/−/Lig4Y288C mice. We demonstrate that, although DSBs do not arise at a high frequency in adult neural stem cells, the low numbers of DSBs that persist endogenously in Lig4Y288C mice or that are induced by low radiation doses can activate apoptosis. A temporal analysis shows that DSB levels in Lig4Y288C mice diminish gradually from the embryo to a steady state level in adult mice. The neonatal SVZ compartment of Lig4Y288C mice harbours diminished DSBs compared to its differentiated counterpart, suggesting a process selecting against unfit stem cells. Finally, we reveal high endogenous apoptosis in the developing SVZ of wild-type newborn mice.

Hot spots of DNA double-strand breaks and genomic contacts of human rDNA units are involved in epigenetic regulation.

DNA double-strand breaks (DSBs) are involved in many cellular mechanisms, including replication, transcription, and genome rearrangements. The recent observation that hot spots of DSBs in human chromosomes delimit DNA domains that possess coordinately expressed genes suggests a strong relationship between the organization of transcription patterns and hot spots of DSBs. In this study, we performed mapping of hot spots of DSBs in a human 43-kb ribosomal DNA (rDNA) repeated unit. We observed that rDNA units corresponded to the most fragile sites in human chromosomes and that these units possessed at least nine specific regions containing clusters of extremely frequently occurring DSBs, which were located exclusively in non-coding intergenic spacer (IGS) regions. The hot spots of DSBs corresponded to only a specific subset of DNase-hypersensitive sites, and coincided with CTCF, PARP1, and HNRNPA2B1 binding sites, and H3K4me3 marks. Our rDNA-4C data indicate that the regions of IGS containing the hot spots of DSBs often form contacts with specific regions in different chromosomes, including the pericentromeric regions, as well as regions that are characterized by H3K27ac and H3K4me3 marks, CTCF binding sites, ChIA-PET and RIP signals, and high levels of DSBs. The data suggest a strong link between chromosome breakage and several different mechanisms of epigenetic regulation of gene expression.

Abstract 4762: Deficient double strand breaks repair of bone marrow plasma cells correlates with better clinical outcome of multiple myeloma patients

Abstract DNA interstrand crosslinks (ICLs) are complex DNA lesions generated by bi-functional alkylating agents, a class of compounds extensively used in cancer chemotherapy. During ICL repair, replication forks stall at the ICL, inducing the formation of a lethal form of DNA damage termed DNA double-strand breaks (DSBs), which are repaired mainly by homologous recombination (HR) and non-homologous end joining (NHEJ). In this study we applied the properties of the ICL-inducing agent melphalan as a model, to elucidate the underlying mechanisms in processing and repair of ICLs. We studied two human multiple myeloma (HMCLs) cell lines (melphalan-sensitive RPMI-8226 and -resistant LR5) and CD138+ bone marrow plasma cells (BMPCs) from 15 newly diagnosed MM patients (8M/7F; median age 61 years) before they receive high-dose melphalan therapy supported by ASCT. HMCLs and BMPCs were treated with melphalan alone or in combination with RI-1 (selective inhibitor of HR) or SCR7 (selective inhibitor of NHEJ) and the extent of the N-ras-specific ICLs using a quantitative PCR assay and DSBs (intermediates of ICL repair) using γH2Ax foci measurements by confocal microscopy were evaluated. The induction of the melphalan-induced apoptosis using a photometric enzyme-assay was also evaluated. The ICLs repair efficiency correlated with response to treatment and could identify 2 groups of patients: non-responders (<PR, n=6), with higher ICLs repair efficiency (t1/2 23h) and responders (≥PR, n=9) with lower efficiency (t1/2 48h). Moreover, using γ-H2AX foci formation/removal measurement, we found that the repair efficiency of DSBs in BMPCs was significantly higher in non-responders (t1/2 9h) than in responders (t1/2 12h) (all p<0.001). Also, the melphalan-induced apoptosis in BMPCs inversely correlated with the repair efficiencies of ICLs and DSBs, with the toxicity being higher in responders than in non-responders. Furthermore, LR5 cells showed higher repair efficiency of both ICLs and DSBs and lower toxicity than RPMI-8226 cells. Interestingly, in all cellular populations analysed, significant correlation between ICL and DSBs levels was observed. To further elucidate the mechanism of drug-induced DSBs repair, HMCLs and BMPCs were treated with melphalan in combination with nontoxic doses of RI-1 or SCR7. We found that the combined treatment of melphalan with RI-1 or SCR7 significantly increased the induction of the melphalan-only phosphorylation of H2AX, delayed the repair of ICLs and strongly enhanced the cytotoxic activity of melphalan (all p<0.01). Collectively, our study demonstrates that significant changes in the repair efficiency of DSBs occur in MM. These changes affect the repair of ICLs, modify drug sensitivity of malignant BMPCs, and correlate with clinical outcome. Specific inhibition of HR and/or NHEJ might be an effective strategy to enhance sensitivity of cancer cells in MM. Citation Format: Maria Gkotzamanidou, Masood Shammas, Evangelos Terpos, Sathees C. Raghavan, Kenneth C. Anderson, Nikhil C. Munshi, Meletios - Athanasios Dimopoulos, Vassilis L. Souliotis. Deficient double strand breaks repair of bone marrow plasma cells correlates with better clinical outcome of multiple myeloma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4762. doi:10.1158/1538-7445.AM2014-4762

DNA breaks and chromosomal aberrations arise when replication meets base excision repair.

Exposures that methylate DNA potently induce DNA double-strand breaks (DSBs) and chromosomal aberrations, which are thought to arise when damaged bases block DNA replication. Here, we demonstrate that DNA methylation damage causes DSB formation when replication interferes with base excision repair (BER), the predominant pathway for repairing methylated bases. We show that cells defective in the N-methylpurine DNA glycosylase, which fail to remove N-methylpurines from DNA and do not initiate BER, display strongly reduced levels of methylation-induced DSBs and chromosomal aberrations compared with wild-type cells. Also, cells unable to generate single-strand breaks (SSBs) at apurinic/apyrimidinic sites do not form DSBs immediately after methylation damage. In contrast, cells deficient in x-ray cross-complementing protein 1, DNA polymerase β, or poly (ADP-ribose) polymerase 1 activity, all of which fail to seal SSBs induced at apurinic/apyrimidinic sites, exhibit strongly elevated levels of methylation-induced DSBs and chromosomal aberrations. We propose that DSBs and chromosomal aberrations after treatment with N-alkylators arise when replication forks collide with SSBs generated during BER.

Repair of ionizing radiation-induced DNA damage and risk of second cancer in childhood cancer survivors

The study's purpose was to assess whether individuals who developed a second malignant neoplasm (SMN) after treatment for a first malignant neoplasm (FMN) had a lower ability to repair DNA double-strand breaks (DSBs) using a bioassay with γH2AX intensity as a surrogate endpoint. In a case-control study nested in a cohort of childhood cancer survivors, lymphoblastoid cell lines (LCLs) were established from blood samples collected from 94 cases (SMN) and 94 matched controls (FMN). LCLs were irradiated with ionizing radiation (2 and 5 Gy) and γH2AX intensities measured 1, 3, 5 and 24h post-irradiation. Differences in mean γH2AX intensity between cases and controls were compared using Kruskal-Wallis tests. Generalized linear models for repeated measures and conditional logistic regressions for SMN risk estimates were performed. The mean baseline γH2AX intensity measured without irradiation was 9.1 [95% confidence interval (95% CI): 8.5-9.7] in the LCLs from cases and 6.4 (95% CI: 6.0-6.8) from controls (P < 0.001). Markedly higher γH2AX intensity, particularly at 1 h post-irradiation, was also found in the LCLs from the cases compared with the controls for all FMNs and for different types of FMN. Chemotherapy and radiation doses received by bone marrow and thymus for FMN treatment showed a non-significant effect on γH2AX intensity. This case-control study shows that higher baseline and post-irradiation levels of DNA DSBs, as measured by γH2AX intensity, are associated with the risk of SMN in childhood cancer survivors. Further investigations in a prospective setting are warranted to confirm this association.

Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts

Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.

Constitutive 53BP1/γH2AX foci are increased in cells of ALL patients dependent on BCR-ABL and TEL-AML1 preleukemic gene fusions.

Childhood leukemia arises from hematopoietic stem cells by induction of mutations. Quite often chromosomal translocations arise prenatally as first key event in multistage process of leukemogenesis. These translocations result in so called preleukemic gene fusions (PGFs), such as BCR-ABL and TEL-AML1, which generate hybrid proteins with altered properties. Critical DNA damage resulting in translocations are DNA double-strand breaks (DSBs). BCR-ABL and TEL-AML1 were shown to be associated with increased constitutive DSBs in various model systems. We analyzed cells from peripheral blood and CD34-/CD34+ cells from bone marrow of pediatric acute lymphoblastic leukemia (ALL) patients harboring BCR-ABL or TEL-AML1. We used sensitive technique that is based on automated enumeration of DSB co-localizing proteins γH2AX and 53BP1, which form so called DNA repair foci. We found that level of constitutive γH2AX/53BP1 foci is significantly higher in cells of ALL pediatric patients than in healthy subjects. There was also significant increased level of constitutive γH2AX/53BP1 foci in cells from ALL patients harboring BCR-ABL or TEL-AML1 compared to patients without PGFs. The same increase was observed regardless cell type for both PGFs. Our data on increased DSB levels in the BCR-ABL/TEL-AML1 patient's cells support a model where BCR-ABL/TEL-AML1 induces DNA instability through facilitating mutagenesis and appearance of additional genetic alterations driving leukemogenesis.

Abstract P4-16-06: Histone deacetylases inhibitor SAHA enhances anti-tumor effects of poly (ADP-ribose) polymerase (PARP) inhibitor olaparib in breast cancer cells

Abstract Background: The poly (ADP-ribose) polymerase (PARP) inhibitor, olaparib, has been found to have a therapeutic potential for treating cancers that have an impaired DNA repair ability. However, some cancer presents acquired resistance to PARP inhibitor or platinum. Histone deacetylases (HDACs) are important to enable functional homologous recombination (HR) by regulating the expression of HR-related genes and promoting the accurate assembly of HR-directed subnuclear foci. Thus, HDAC inhibitors have emerged recently as a class of therapeutic agents for the treatment of cancer by inhibiting DNA repair. For this mechanism, HDAC inhibition would enhance the anti-tumor effect of PARP inhibitor in cancer cells by blocking DNA repair pathway. Materials and Methods: We determined whether SAHA, a HDAC inhibitor could enhance the growth inhibition of olaparib on breast cancer cell lines using MTT assay. We examined whether exposure to SAHA affects the expression level of genes involved in HR. The accumulation of DNA double strand breaks (DSBs) induced by combination treatment was accessed by the comet assay. Cell cycle analysis and molecular changes induced by combination of olaparib plus SAHA were also performed. These in vitro data were validated in the in vivo xenograft model as well. Results: Triple-negative breast cancer cell lines showed heterogeneous response to dual inhibition of PARP and HDACs. SAHA enhanced olaparib-induced cell death of MDA-MB-157 and HCC1143 but not of HCC70 and MDA-MB-468. Combination of olaparib plus SAHA caused a greater decrease of pAKT, pERK, and pSTAT3 in MDA-MB-157 and HCC1143 cells compared with monotherapy either olaparib or SAHA. Furthermore, inhibition of PARP increased the accumulation of DNA DSBs induced by SAHA in these two cell lines, MDA-MB-157 and HCC1143. There was no change in proliferative pathway activation in HCC70 and MDA-MB-468 cells with combination of olaparib plus SAHA. Our findings showed that triple-negative breast cancer cells are differentially effective to combination of SAHA plus olaparib which increased levels of unrepaired DNA DSBs. Conclusion: HDAC inhibitor SAHA enhances growth inhibitory activity of PARP inhibitor olaparib in several triple-negative breast cancer cells with increased accumulation of DNA DSBs. Our results provide a rationale for the future clinical trials of olaparib combined with SAHA in the treatment of cancers that have an impaired DNA repair ability. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-16-06.

The DNA double-strand break response is abnormal in myeloblasts from patients with therapy-related acute myeloid leukemia

The complex chromosomal aberrations found in therapy related acute myeloid leukemia (t-AML) suggest that the DNA double strand break (DSB) response may be altered. In this study we examined the DNA DSB response of primary bone marrow cells from t-AML patients and performed next-generation sequencing of 37 canonical homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA repair genes, and a subset of DNA damage response genes using tumor and paired normal DNA obtained from t-AML patients. Our results suggest that the majority of t-AML patients (11 of 15) have tumor cell-intrinsic, functional dysregulation of their DSB response. Distinct patterns of abnormal DNA damage response in myeloblasts correlated with acquired genetic alterations in TP53 and the presence of inferred chromothripsis. Furthermore, the presence of trisomy 8 in tumor cells was associated with persistently elevated levels of DSBs. Although tumor-acquired point mutations or small indels in canonical HR and NHEJ genes do not appear to be a dominant means by which t-AML leukemogenesis occurs, our functional studies suggest that an abnormal response to DNA damage is a common finding in t-AML.