Elevated Levels Of DNA Damage Research Articles

Elevated Level of DNA Damage and Impaired Repair of DNA Oxidative Damage in Multiple Sclerosis Patients (P7-6.010)

Elevated Level of DNA Damage and Impaired Repair of DNA Oxidative Damage in Multiple Sclerosis Patients (P7-6.010)

Serum Amyloid A3 Fuels a Feed-Forward Inflammatory Response to the Bacterial Amyloid Curli in the Enteric Nervous System

BackgroundMounting evidence suggests a role for the gastro-intestinal microbiome as a determinant of peripheral immunity and central neurodegeneration, but the local disease mechanisms remain unknown. Given its potential relevance for early diagnosis and therapeutic intervention, we set out to map the pathogenic changes induced by bacterial amyloids in the gastro-intestinal tract and its enteric nervous system. MethodsTo examine the early response, we challenged primary murine myenteric networks with curli, the prototypic bacterial amyloid, and performed shotgun RNA sequencing and multiplex ELISA. Using enteric neurosphere-derived glial and neuronal cell cultures, as well as in vivo curli injections into the colon wall, we further scrutinized curli-induced pathogenic pathways. ResultsCurli induced a pro-inflammatory response, with marked upregulation of Serum Amyloid A3 (Saa3) and the secretion of several cytokines. This pro-inflammatory state was primarily induced in enteric glia, was accompanied by elevated levels of DNA damage and replication, and triggered the influx of immune cells in vivo. The addition of recombinant SAA3 was sufficient to recapitulate this specific pro-inflammatory phenotype while Saa3 knock-out attenuated curli-induced DNA damage and replication. Like curli, recombinant SAA3 caused a strong upregulation of Saa3 transcripts, indicating a feed-forward loop. Colonization of curli-producing Salmonella and Dextran Sulphate Sodium (DSS)-induced colitis caused a significant increase in Saa3 transcripts, indicating a central role for SAA3 in enteric dysfunction. Inhibition of dual leucine zipper kinase (DLK), an upstream regulator of the c-Jun N-terminal kinase (JNK) pathway responsible for SAA3 production, attenuated curli- and SAA3-induced Saa3 upregulation, DNA damage and replication in enteric glia. ConclusionsOur results position SAA3 as an important mediator of gastro-intestinal vulnerability towards bacterial-derived amyloids and demonstrate the potential of DLK inhibition to dampen enteric pathology.

#289 : Mitochondrial Activator BGP-15 Protects Sperm from DNA Damage During Sperm Recovery for Assisted Reproductive Technology

Background and Aims: The recovery of sperm by assisted reproductive technology (ART) results in significant DNA damage. Elevated levels of DNA damage in sperm have been linked to poor-quality embryos and increased pregnancy loss after ART. Therefore, in this study, we aimed to investigate potential of the new mitochondrial activator BGP-15 to prevent sperm DNA damage in an in vitroejaculate over time. Additionally, we sought to determine whether BGP-15 can preserve sperm quality during various sperm recovery techniques used in ART. Method: We incubated donated human semen specimens untreated or treated with BGP-15, then examined sperm motility and DNA damage (DNA fragmentation and oxidation). Semen samples were then processed using different clinical sperm recovery methods: simple wash (SW), swim-up (SU) and density gradient centrifugation (DGC); and the purified sperm underwent further analysis including motility, vitality, ROS levels, mitochondrial membrane integrity and mitochondrial membrane potential (MMP), and DNA damage. Results: Semen samples incubated with BGP-15 demonstrated improved sperm motility and reduced DNA damage levels. SW samples had the highest sperm count, while SU had the highest vitality and DNA integrity, but the lowest MMP. DGC sperm had increased MMP after BGP-15 Treatment. Although BGP-15 treatment only reduced sperm DNA fragmentation in SW samples, it reduced DNA oxidation in all recovery methods. Conclusion: Our findings indicate that clinical manipulation of patient semen samples can negatively impact sperm quality, and different sperm recovery methods have unique effects. However, the addition of BGP-15 to clinical processes can preserve sperm quality, potentially leading to improved embryo quality and ART success.

Lineage skewing and genome instability underlie marrow failure in a zebrafish model of GATA2 deficiency

Inherited bone marrow failure associated with heterozygous mutations in GATA2 predisposes toward hematological malignancies, but the mechanisms remain poorly understood. Here, we investigate the mechanistic basis of marrow failure in a zebrafish model of GATA2 deficiency. Single-cell transcriptomics and chromatin accessibility assays reveal that loss of gata2a leads to skewing toward the erythroid lineage at the expense of myeloid cells, associated with loss of cebpa expression and decreased PU.1 and CEBPA transcription factor accessibility in hematopoietic stem and progenitor cells (HSPCs). Furthermore, gata2a mutants show impaired expression of npm1a, the zebrafish NPM1 ortholog. Progressive loss of npm1a in HSPCs is associated with elevated levels of DNA damage in gata2a mutants. Thus, Gata2a maintains myeloid lineage priming through cebpa and protects against genome instability and marrow failure by maintaining expression of npm1a. Our results establish a potential mechanism underlying bone marrow failure in GATA2 deficiency.

Abstract 5496: Glutaminase inhibition induces replication stress in ovarian cancer cells and inhibition of replication checkpoint causes synthetic lethality

Abstract Ovarian cancer (OC) is a highly aggressive disease and the most lethal gynecologic malignancy in women. Although the majority of OC patients respond to chemotherapeutic drugs, more than 70% of the patients relapse and die as a result of chemoresistance. Therefore, novel therapeutics are warranted to prevent chemoresistance and treat the relapsed disease to improve prognosis. Many cancer cells depend on Glutamine as major carbon source and these cells have high Glutaminase (GLS) expression, an enzyme that converts Glutamine to Glutamate. Chemo-resistant OC cells have elevated levels of GLS, which confirms their increased dependency on glutamine metabolism. Based on these observations, we postulated that GLS inhibition may attenuate the aggressive growth of GLS high OC cells and may sensitize to the agents that can further potentiate these effects. Interestingly, GLS inhibition using a clinical-stage drug CB839 caused replication stress and activated DNA damage checkpoint protein 1 (CHK1) mediated cell cycle arrest. These novel findings suggested a role for CHK1 in protecting GLS inhibition-induced DNA damage by facilitating the timely repair of DNA breaks. Based on these observations, we hypothesized that GLS inhibition in combination with CHK1 inhibition may cause synergistic lethality in chemo-resistant and GLShigh OC cells. We evaluated the combination of GLS inhibitor CB839 and CHK1 inhibitor Prexasertib. Our results demonstrate that GLS inhibition-induced CHK1 phosphorylation is significantly attenuated by Prexasertib treatment. Similarly, combined treatment of CB839 and Prexasertib showed significantly elevated levels of DNA damage as measured by COMET assays, replication stress-mediated DNA damage responses, and synergistic OC cell lethality compared to individual drug treatments. Furthermore, CB839 and Prexasertib combination was more synergistic in cells that expressed high GLS compared to low GLS-expressing OC cells indicating the specificity of the combination of these drugs. Together, our studies identified a novel connection between metabolic and DNA damage checkpoint pathways in OC and propose a novel synergistic lethality-based combination therapy to treat chemo-resistant and aggressive OC. Citation Format: Pamela Luna, Ganesh Acharya, Damieanus Ochola, Swetha Peddibhotla, Chinnadurai Mani, Mark B. Reedy, Komaraiah Palle. Glutaminase inhibition induces replication stress in ovarian cancer cells and inhibition of replication checkpoint causes synthetic lethality. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5496.

Analysis of Single- and Double-Stranded DNA Damage in Osteoblastic Cells after Hyperbaric Oxygen Exposure.

(1) Background: Hyperbaric oxygen (HBO) exposure induces oxidative stress that may lead to DNA damage, which has been observed in human peripheral blood lymphocytes or non-human cells. Here, we investigated the impact of hyperbaric conditions on two human osteoblastic cell lines: primary human osteoblasts, HOBs, and the osteogenic tumor cell line SAOS-2. (2) Methods: Cells were exposed to HBO in an experimental hyperbaric chamber (4 ATA, 100% oxygen, 37 °C, and 4 h) or sham-exposed (1 ATA, air, 37 °C, and 4 h). DNA damage was examined before, directly after, and 24 h after exposure with an alkaline comet assay and detection of γH2AX+53BP1 colocalizing double-strand break (DSB) foci and apoptosis. The gene expression of TGFß-1, HO-1, and NQO1, involved in antioxidative functions, was measured with qRT-PCR. (3) Results: The alkaline comet assay showed significantly elevated levels of DNA damage in both cell lines after 4 h of HBO, while the DSB foci were similar to sham. γH2AX analysis indicated a slight increase in apoptosis in both cell lines. The increased expression of HO-1 in HOB and SAOS-2 directly after exposure suggested the induction of an antioxidative response in these cells. Additionally, the expression of TGF-ß1 was negatively affected in HOB cells 4 h after exposure. (4) Conclusions: in summary, this study indicates that osteoblastic cells are sensitive to the DNA-damaging effects of hyperbaric hyperoxia, with the HBO-induced DNA damage consisting largely of single-strand DNA breaks that are rapidly repaired.

Combined Approach: FFQ, DII, Anthropometric, Biochemical and DNA Damage Parameters in Obese with BMI ≥ 35 kg m-2.

Although obesity with its comorbidities is linked with higher cancer risk, the data on genome stability in the obese/severely obese are scarce. This is the first study with three DNA damage assessment assays (Fpg-modified and alkaline comet assays and micronucleus cytome assay) performed on a severely obese population (n = 53) where the results were compared with daily intake of food groups, nutrient intake, dietary inflammatory index (DII), and anthropometric and biochemical parameters usually measured in obese individuals. Results demonstrated the association between DNA damage levels and a decrease in cell proliferation with anthropometric measurements and the severity of obese status, together with elevated levels of urates, inorganic phosphates, chlorides, and hs troponin I levels. DII was connected with oxidative DNA damage, while BMI and basal metabolic rate (BMR) were associated with a decrease in cell proliferation and DNA damage creation. Measured daily BMR and calculated daily energy intake from the food frequency questionnaire (FFQ) demonstrated no significant difference (1792.80 vs. 1869.86 kcal day-1 mean values). Groups with higher DNA damage than expected (tail intensity in comet assay >9% and >12.4%, micronucleus frequency >13), consumed daily, weekly, and monthly more often some type of food groups, but differences did not show a clear influence on the elevated DNA damage levels. Combination of all three DNA damage assays demonstrated that some type of damage can start earlier in the obese individual lifespan, such as nuclear buds and nucleoplasmic bridges, then comes decrease in cell proliferation and then elevated micronucleus frequencies, and that primary DNA damage is not maybe crucial in the overweight, but in severely obese. Biochemically changed parameters pointed out that obesity can have an impact on changes in blood cell counts and division and also on genomic instability. Assays were able to demonstrate groups of sensitive individuals that should be further monitored for genomic instability and cancer prevention, especially when obesity is already connected with comorbidities, 13 different cancers, and a higher mortality risk with 7-10 disease-free years loss. In the future, both DNA damage and biochemical parameters should be combined with anthropometric ones for further obese monitoring, better insight into biological changes in the severely obese, and a more individual approach in therapy and treatment. Patients should also get a proper education about the foodstuff with pro- and anti-inflammatory effect.

Incorporation of 53BP1 into phase-separated bodies in cancer cells during aberrant mitosis.

53BP1 (also known as TP53BP1) is a key mediator of the non-homologous end joining (NHEJ) DNA repair pathway, which is the primary repair pathway in interphase cells. However, the mitotic functions of 53BP1 are less well understood. Here, we describe 53BP1 mitotic stress bodies (MSBs) formed in cancer cell lines in response to delayed mitosis. These bodies displayed liquid-liquid phase separation characteristics, were close to centromeres, and included lamin A/C and the DNA repair protein RIF1. After release from mitotic arrest, 53BP1 MSBs decreased in number and moved away from the chromatin. Using GFP fusion constructs, we found that the 53BP1 oligomerization domain region was required for MSB formation, and that inclusion of the 53BP1 N terminus increased MSB size. Exogenous expression of 53BP1 did not increase MSB size or number but did increase levels of MSB-free 53BP1. This was associated with slower mitotic progression, elevated levels of DNA damage and increased apoptosis, which is consistent with MSBs suppressing a mitotic surveillance by 53BP1 through sequestration. The 53BP1 MSBs, which were also found spontaneously in a subset of normally dividing cancer cells but not in non-transformed cells (ARPE-19), might facilitate the survival of cancer cells following aberrant mitoses. This article has an associated First Person interview with the first author of the paper.

Oxidative Stress and DNA Damage in Peripheral Blood Mononuclear Cells from Normal, Obese, Prediabetic and Diabetic Persons Exposed to Thyroid Hormone In Vitro.

Diabetes, a chronic group of medical disorders characterized byhyperglycemia, has become a global pandemic. Some hormones may influence the course and outcome of diabetes, especially if they potentiate the formation of reactive oxygen species (ROS). There is a close relationship between thyroid disorders and diabetes. The main objective of this investigation was to find out whether peripheral blood mononuclear cells (PBMCs) are more prone to DNA damage by triiodothyronine (T3) (0.1, 1 and 10 μM) at various stages of progression through diabetes (obese, prediabetics, and type 2 diabetes mellitus—T2DM persons). In addition, some biochemical parameters of oxidative stress (catalase-CAT, thiobarbituric acid reactive substances—TBARS) and lactate dehydrogenase (LDH) were evaluated. PBMCs from prediabetic and diabetic patients exhibited increased sensitivity for T3 regarding elevated level of DNA damage, inhibition of catalase, and increase of TBARS and LDH. PBMCs from obese patients reacted in the same manner, except for DNA damage. The results of this study should contribute to a better understanding of the role of thyroid hormones in the progression of T2DM.

Reduced nuclear NAD+ drives DNA damage and subsequent immune activation in the retina.

Mutations in NMNAT1, a key enzyme involved in the synthesis of NAD+ in the nucleus, lead to an early onset severe inherited retinal degeneration (IRD). We aimed to understand the role of nuclear NAD+ in the retina and to identify the molecular mechanisms underlying NMNAT1-associated disease, using a mouse model that harbors the p.V9M mutation in Nmnat1 (Nmnat1V9M/V9M). We identified temporal transcriptional reprogramming in the retinas of Nmnat1V9M/V9M mice prior to retinal degeneration, which begins at 4weeks of age, with no significant alterations in gene expression at 2weeks of age and over 2600 differentially expressed genes by 3weeks of age. Expression of the primary consumer of NAD+ in the nucleus, PARP1, an enzyme involved in DNA damage repair and transcriptional regulation, as well as 7 other PARP family enzymes, was elevated in the retinas of Nmnat1V9M/V9M. This was associated with elevated levels of DNA damage, PARP-mediated NAD+ consumption and migration of Iba1+/CD45+ microglia/macrophages to the subretinal space in the retinas of Nmnat1V9M/V9M mice. These findings suggest that photoreceptor cells are especially sensitive to perturbation of genome homeostasis, and that PARP-mediated cell death may play a role in other genetic forms of IRDs, and potentially other forms of neurodegeneration.

Methylglyoxal induces chromosomal instability and mitotic dysfunction in lymphocytes.

Type 2 diabetes is associated with elevated levels of DNA damage, in particular micronuclei (MNi) which are formed by acentric chromosome fragments caused by double-stranded DNA breaks (DSBs), or whole chromosomes which fail to segregate during mitosis. We investigated if methylglyoxal (MGO), a reactive dicarbonyl known to be elevated in type 2 diabetes is capable of increasing chromosomal instability and DNA damage as measured by the cytokinesis block micronucleus cytome (CBMNcyt) assay in B-lymphoblastoid WIL2-NS cells and primary peripheral blood lymphocytes (PBL). We also investigated the level of various dicarbonyl stress biomarkers, including extracellular and intracellular MGO, protein and MGO modifications of DNA. WIL2-NS cells exposed to either MGO or a glyoxalase 1 inhibitor showed increases in MNi and nuclear buds, which were associated with an increase in intracellular MGO. DNA damage in the form of MNi and nucleoplasmic bridges were observed in primary PBL exposed to 10 µM MGO, suggesting low concentrations of MGO may be genotoxic. Furthermore, we showed, using fluorescent in situ hybridisation, that the majority of MNi caused by MGO in WIL2-NS cells were caused by whole chromosome loss events, rather than DSBs. Our data suggest that MGO, a reactive metabolite elevated in type 2 diabetes and other pathologies, can affect genomic integrity by impairing chromosome segregation during mitosis.

Real-world particulate matters induce lung toxicity in rats fed with a high-fat diet: Evidence of histone modifications

Exposure to ambient particulate matters (PMs) has been associated with a variety of lung diseases, and high-fat diet (HFD) was reported to exacerbate PM-induced lung dysfunction. However, the underlying mechanisms for the combined effects of HFD and PM on lung functions remain poorly unraveled. By performing a comparative proteomic analysis, the current study investigated the global changes of histone post-translational modifications (PTMs) in rat lung exposed to long-term, real-world PMs. In result, after PM exposure the abundance of four individual histone PTMs (1 down-regulated and 3 up-regulated) and six combinatorial PTMs (1 down-regulated and 5 up-regulated) were significantly altered in HFD-fed rats while only one individual PTM was changed in rats with normal diet (ND) feeding. Histones H3K18ac, H4K8ac and H4K12ac were reported to be associated with DNA damage response, and we found that these PTMs were enhanced by PM in HFD-fed rats. Together with the elevated DNA damage levels in rat lungs following PM and HFD co-exposure, we demonstrate that PM exposure combined with HFD could induce lung injury through altering more histone modifications accompanied by DNA damage. Overall, these findings will augment our knowledge of the epigenetic mechanisms for pulmonary toxicity caused by ambient PM and HFD exposure.

MYBL2 and ATM suppress replication stress in pluripotent stem cells

Replication stress, a major cause of genome instability in cycling cells, is mainly prevented by the ATR‐dependent replication stress response pathway in somatic cells. However, the replication stress response pathway in embryonic stem cells (ESCs) may be different due to alterations in cell cycle phase length. The transcription factor MYBL2, which is implicated in cell cycle regulation, is expressed a hundred to a thousand‐fold more in ESCs compared with somatic cells. Here we show that MYBL2 activates ATM and suppresses replication stress in ESCs. Consequently, loss of MYBL2 or inhibition of ATM or Mre11 in ESCs results in replication fork slowing, increased fork stalling and elevated origin firing. Additionally, we demonstrate that inhibition of CDC7 activity rescues replication stress induced by MYBL2 loss and ATM inhibition, suggesting that uncontrolled new origin firing may underlie the replication stress phenotype resulting from loss/inhibition of MYBL2 and ATM. Overall, our study proposes that in addition to ATR, a MYBL2‐MRN‐ATM replication stress response pathway functions in ESCs to control DNA replication initiation and prevent genome instability.

Telomere shortening and DNA damage in culprit cells of different types of progressive fibrosing interstitial lung disease.

Pulmonary fibrosis is strongly associated with telomere shortening and increased DNA damage. Key cells in the pathogenesis involve alveolar type 2 (AT2) cells, club cells and myofibroblasts; however, to what extent these cells are affected by telomere shortening and DNA damage is not yet known. We sought to determine the degree of, and correlation between, telomere shortening and DNA damage in different cell types involved in the pathogenesis of progressive fibrosing interstitial lung disease. Telomere length and DNA damage were quantified, using combined fluorescence in situ hybridisation and immunofluorescence staining techniques, in AT2 cells, club cells and myofibroblasts of controls and patients with pulmonary fibrosis and a telomerase reverse transcriptase mutation (TERT-PF), idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (fHP). In IPF and TERT-PF lungs, AT2 cells contained shorter telomeres and expressed higher DNA damage signals than club cells and myofibroblasts. In fHP lungs, club cells contained highly elevated levels of DNA damage, while telomeres were not obviously short. In vitro, we found significantly shorter telomeres and higher DNA damage levels only in AT2 surrogate cell lines treated with telomerase inhibitor BIBR1532. Our study demonstrated that in IPF and TERT-PF lungs, telomere shortening and accumulation of DNA damage primarily affects AT2 cells, further supporting the importance of AT2 cells in these diseases, while in fHP the particularly high telomere-independent DNA damage signals in club cells underscores its bronchiolocentric pathogenesis. These findings suggest that cell type-specific telomere shortening and DNA damage may help to discriminate between different drivers of fibrogenesis.

Replication stress signaling is a therapeutic target in myelodysplastic syndromes with splicing factor mutations.

Somatic mutations in genes coding for splicing factors, e.g., SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with myelodysplastic syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease, therefore, making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation- associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with the low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association between R-loops and ATR sensitivity and the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.

Abstract 2371: FBXL12 modulates Fanconi anaemia-BRCA signaling under conditions of oncogene-induced replication stress

Abstract Oncogene-induced replication stress (OIRS) constitutes an early obstacle to cancer progression. De-regulation of cell death or senescence programs contribute to overcoming this barrier. However, even after escaping these fail-safe mechanisms, cancer cells need to handle elevated levels of DNA damage and ensuing genomic instability in order to continue proliferating. How this is achieved is only partially understood. To advance our understanding of this response and to identify potentially druggable regulators, we screened the family of human F-box proteins (FBP) for effects on replication re-start under conditions of OIRS. FBPs function as interchangeable substrate recognition sub-units of the SKP1-Cullin1-FBP ubiquitin ligase complex. They are required for conferring substrate specificity to the complex and some FBPs have been attributed roles as oncogenes or tumor suppressors while the majority remain largely uncharacterized. Our screen revealed the relatively unknown FBP FBXL12 as crucial regulator for sustained proliferation and genomic integrity under conditions of cyclin E-induced replication stress. FBXL12-depleted cells exhibit excessive replication fork stalling, high levels of DNA damage signaling and spontaneous senescence in OIRS breast cancer model cell lines. Interestingly, the same effects are not manifest in untransformed cell lines. Furthermore, we found that FBXL12 interacts with central chromatin-associated components of the Fanconi anemia (FA)-BRCA signaling pathway, ubiquitylating and targeting them for proteasomal degradation, and thereby exerting its effects on stalled replication forks. The specific nature of FA-BRCA signaling regulation by FBXL12 as well as potential clinical implications for basal-like breast cancers will be discussed. Citation Format: Andrä Brunner, Henrik Johansson, Alexandros Kourtesakis, Juha Rantala, Charles Spruck, James Wohlschlegel, Janne Lehtiö, Olle Sangfelt. FBXL12 modulates Fanconi anaemia-BRCA signaling under conditions of oncogene-induced replication stress [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2371.

Thymidylate synthase inhibitor raltitrexed can induce high levels of DNA damage in MYCN-amplified neuroblastoma cells.

MYCN gene amplification is consistently associated with poor prognosis in patients with neuroblastoma, a pediatric tumor arising from the sympathetic nervous system. Conventional anticancer drugs, such as alkylating agents and platinum compounds, have been used for the treatment of high‐risk patients with MYCN‐amplified neuroblastoma, whereas molecule‐targeting drugs have not yet been approved. Therefore, the development of a safe and effective therapeutic approach is highly desired. Although thymidylate synthase inhibitors are widely used for colorectal and gastric cancers, their usefulness in neuroblastoma has not been well studied. Here, we investigated the efficacies of approved antifolates, methotrexate, pemetrexed, and raltitrexed (RTX), on MYCN‐amplified and nonamplified neuroblastoma cell lines. Cell growth‐inhibitory assay revealed that RTX showed a superior inhibitory activity against MYCN‐amplified cell lines. We found no significant differences in the protein expression levels of the antifolate transporter or thymidylate synthase, a primary target of RTX, among the cell lines. Because thymidine supplementation could rescue the RTX‐induced cell growth suppression, the effect of RTX was mainly due to the reduction in dTTP synthesis. Interestingly, RTX treatments induced single‐stranded DNA damage response in MYCN‐amplified cells to a greater extent than in the nonamplified cells. We propose that the high DNA replication stress and elevated levels of DNA damage, which are a result of deregulated expression of MYCN target genes, could be the cause of increased sensitivity to RTX.

Enhanced apoptogenesis and oncogene regulatory mechanism of troxerutin in triple negative breast cancer cells.

Triple negative breast carcinoma (TNBC) is an aggressive form of cancer, with high rates of morbidity, mortality, poor prognosis and limited therapeutic options. The objective of the present study was to elaborate the anticancer activity of Troxerutin (TXN) in TNBC/MDA-MB-231 cells. Herein, we demonstrated the inhibitory effects of TXN on the breast cancer cell growth via induction of apoptosis. Mitochondrial membrane potential (∆Ψm), DNA damage and apoptotic nuclear changes were analyzed by flowcytometry, AO/EtBr and Hoechst staining, respectively. Furthermore, apoptotic protein and gene expressions were analyzed by western blot and reverse transcription polymerase chain reaction (RT-PCR), respectively. Our results indicated that TXN induces apoptosis as evidenced by inhibit the cell proliferation, enhanced apoptotic activation, altered mitochondrial membrane potential and elevated level of DNA damage in TNBC cells. Furthermore, the TXN inhibit anti-apoptotic protein expression with the subsequent upregulation of Cytochrome c, Caspase-9 and Caspase-3. Thus, TXN induces apoptosis in TNBC cells through inducing nuclear damage and altered apoptotic marker expressions. Therefore, TXN might be used as a potential therapeutic agent for the treatment of triple negative breast cancer.

Acetaminophen‐induced Liver Injury Induces DNA Damage and Activation of Ataxia Telangiectasia Mutated in Mice

Acetaminophen (APAP) is a common analgesic/antipyretic that is widely used throughout the world. At therapeutic doses it is safe but at higher dosages APAP causes acute liver failure (ALF). Drug‐induced liver toxicity is the most common cause of liver damage, accounting for 50% of the cases of liver failure. With the only efficacious form of treatment being liver transplant, it is imperative to find alternative forms of treatment. Gaining a greater understanding of the molecular pathology contributing to the progression of liver failure, we allow insights on methods to enhance patient care and quality of life. Acute liver injury from APAP is associated with increased levels of inflammation, oxidative stress, and hepatic cell death. High levels of oxidative stress damages multiple systems within the cell, most notability the DNA. In response to DNA damage, DNA damage response (DDR) markers are activated leading to cell cycle arrest, DNA repair and the eventual death of the cell if damage is unable to be repaired. Our hypothesis is that the accumulation of DNA damage results in the activation of the DDR, which then aids in driving further progression of APAP‐induced liver injury.MethodsC57Bl/6 mice were injected with APAP (300mg/kg) do induce liver injury. Liver tissue was collected after 1, 2, 4, 6, 12, and 24 hours. Histology was performed on paraffin‐embedded sections using antibodies specific for phospho‐ATM (ser 1981) and γH2AX (ser 139). Replication protein A (RPA), Ataxia Telangiectasia mutated (ATM) and 53BP were assessed by qPCR. Phospho‐ATM and RPA were assessed by immunoblotting.ResultsAPAP injected mice showed a marked increase in the accumulation of DNA damage when compared to saline injected mice in the liver. Markers for DNA double strand breaks (DSB), γH2AX and 53BP, were shown to be elevated 2 hours after APAP injection followed by a gradual decrease. RPA, a single stranded DNA‐binding protein, was also shown to be elevated 4 hours after APAP injection indicating an accumulation of single strand breaks. ATM, a DDR maker, and the phosphorylated form of ATM were shown to also be elevated after APAP injection after onset of DSB, with peak expression at 4 hours.ConclusionDuring APAP‐induced liver injury, there is an elevated level of DNA damage found within the liver. This elevated amount of DNA damage results in the activation of ATM and the DDR. ATM‐mediated signaling may be a potential driver for hepatocellular death and ALF. Agents designed to inhibit ATM‐mediated signaling or the DDR response need to be assessed as potential therapeutic targets for the treatment of APAP‐induced liver damage.Support or Funding InformationThis study was funded by NIH R01 awards (DK082435 and DK112803) and a VA Merit award (BX002638) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. DeMorrow. This work was completed with support from the Veterans Health Administration and with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

P5434P300-mediated inactivation of p53 protects against doxorubicin-induced cardiotoxicity

Abstract Introduction Doxorubicin is amongst the most widely prescribed chemotherapy drugs due to its effectiveness in cancer treatment. However, progressive treatment using doxorubicin severely increases the risk of congestive heart failure. Mechanistically, researchers have shown that doxorubicin (i) intercalates into DNA and disrupts topoisomerase-II-mediated DNA repair and (ii) generates free radicals which then causes damage to cellular membranes, DNA, and proteins. Ultimately these cellular insults induce cardiomyocyte (CM) death mediated by the tumour suppressor, p53. Currently, there are no clinically applicable preventative treatments for doxorubicin-induced cardiotoxicity and so, extensive research is being done in discovering a potential therapy. One such candidate is curcumin – a natural polyphenol compound non-toxic to humans. We have previously demonstrated that curcumin inhibits lysine acetyltransferase activity of p300, therefore reducing both histone and non-histone protein acetylation. To induce CM death, p53 requires acetylation by p300. Therefore, we hypothesize that curcumin protects against doxorubicin-induced CM death and cardiotoxicity via p300-mediated inactivation of p53. Methods Rat H9c2 cardiomyoblast cells were cultured and treated with a 2.5 μM dose of doxorubicin for 16 hours. One group of cells were pre-treated with curcumin (15μM) 4 hours prior to doxorubicin treatment, and controls were cultured with only diluent added. Following treatment, the cells were harvested for total protein. At end point, we performed immunoblotting to measure protein expression of key proteins involved in DNA damage (y-H2A.X, p53), and apoptosis (cleaved-Caspase 3). Results Our findings show that following doxorubicin treatment, p53 expression was significantly increased (p<0.001), confirming its role in doxorubicin-associated cardiotoxicity. Furthermore, doxorubicin was associated with increased DNA-damage as evidenced by increased expression and activation of DNA double-stranded breaks (DSBs) marker, y-H2A.X (p<0.001). Elevated levels of DNA-damage were further associated with significantly increased doxorubicin-induced apoptosis as measured by immunoblotting for cleaved-Caspase 3 (p<0.001). However, DNA-damage and apoptosis were attenuated by pre-treatment of cells with curcumin. Curcumin treatment led to a significant decrease in both y-H2A.X (p<0.01) and cleaved-Caspase 3 (p<0.01) expression compared to cells treated with only doxorubicin. Conclusions Our data provides the first evidence that curcumin protects against doxorubicin-induced apoptosis in rat cardiomyoblast cells in vitro. Further studies are warranted in vivo to confirm the potential of curcumin as a cardio-protective drug. Curcumin is a natural compound with little to no side-effects in humans, therefore our finding may provide a novel therapeutic target and treatment approach for doxorubicin-associated cardiotoxicity. Acknowledgement/Funding Heart & Stroke Foundation of Canada