Neutral Comet Assay Research Articles

Norepinephrine-Induced DNA Damage in Ovarian Cancer Cells.

Multiple studies have shown that psychological distress in epithelial ovarian cancer (EOC) patients is associated with worse quality of life and poor treatment adherence. This may influence chemotherapy response and prognosis. Moreover, although stress hormones can reduce cisplatin efficacy in EOC treatment, their effect on the integrity of DNA remains poorly understood. In this study, we investigated whether norepinephrine and epinephrine can induce DNA damage and modulate cisplatin-induced DNA damage in three EOC cell lines. Our data show that norepinephrine and epinephrine exposure led to increased nuclear γ-H2AX foci formation in EOC cells, a marker of double-strand DNA breaks. We further characterized norepinephrine-induced DNA damage by subjecting EOC cells to alkaline and neutral comet assays. Norepinephrine exposure caused DNA double-strand breaks, but not single-strand breaks. Interestingly, pre-treatment with propranolol abrogated norepinephrine-induced DNA damage indicating that its effects may be mediated by β-adrenergic receptors. Lastly, we determined the effects of norepinephrine on cisplatin-induced DNA damage. Our data suggest that norepinephrine reduced cisplatin-induced DNA damage in EOC cells and that this effect may be mediated independently of β-adrenergic receptors. Taken together, these results suggest that stress hormones can affect DNA integrity and modulate cisplatin resistance in EOC cells.

EGFR Amplification Induces Increased DNA Damage Response and Renders Selective Sensitivity to Talazoparib (PARP Inhibitor) in Glioblastoma.

Exploration of novel strategies to extend the benefit of PARP inhibitors beyond BRCA-mutant cancers is of great interest in personalized medicine. Here, we identified EGFR amplification as a potential biomarker to predict sensitivity to PARP inhibition, providing selection for the glioblastoma (GBM) patient population who will benefit from PARP inhibition therapy. Selective sensitivity to the PARP inhibitor talazoparib was screened and validated in two sets [test set (n = 14) and validation set (n = 13)] of well-characterized patient-derived glioma sphere-forming cells (GSC). FISH was used to detect EGFR copy number. DNA damage response following talazoparib treatment was evaluated by γH2AX and 53BP1 staining and neutral comet assay. PARP-DNA trapping was analyzed by subcellular fractionation. The selective monotherapy of talazoparib was confirmed using in vivo glioma models. EGFR-amplified GSCs showed remarkable sensitivity to talazoparib treatment. EGFR amplification was associated with increased reactive oxygen species (ROS) and subsequent increased basal expression of DNA-repair pathways to counterelevated oxidative stress, and thus rendered vulnerability to PARP inhibition. Following talazoparib treatment, EGFR-amplified GSCs showed enhanced DNA damage and increased PARP-DNA trapping, which augmented the cytotoxicity. EGFR amplification-associated selective sensitivity was further supported by the in vivo experimental results showing that talazoparib significantly suppressed tumor growth in EGFR-amplified subcutaneous models but not in nonamplified models. EGFR-amplified cells are highly sensitive to talazoparib. Our data provide insight into the potential of using EGFR amplification as a selection biomarker for the development of personalized therapy.

Let-7e Suppresses DNA Damage Repair and Sensitizes Ovarian Cancer to Cisplatin through Targeting PARP1.

Increased DNA damage repair is one of the mechanisms implicated in cisplatin resistance. Our previous study indicated that the deregulation of let-7e promoted cisplatin resistance and that let-7e could suppress DNA double-strand break repair in ovarian cancer. In this study, we further characterized the role of let-7e in DNA damage repair and cisplatin resistance in ovarian cancer, and investigated the underlying mechanisms. The alkaline and neutral comet assay indicated that let-7e impeded both DNA single- and double-strand break repairs through downregulating its target gene PARP1. In vitro and in vivo experiments provided evidence that the let-7e-PARP1-DNA repair axis was involved in the modulation of cisplatin sensitivity in ovarian cancer. Contrary to let-7e, PARP1 was overexpressed in cisplatin-resistant ovarian cancer tissues, and patients with high PARP1 expression exhibited poor progression-free survival (PFS) and overall survival (OS). Multivariate logistic and Cox regression analyses showed that let-7e and FIGO stage were independent prognostic factors for PFS and OS, whereas let-7e and PARP1 were able to independently predict chemotherapy response. Taken together, our results indicated that low expression of let-7e promoted DNA single- and double-strand break repairs and subsequently contributed to cisplatin resistance by relieving the suppression on PARP1 in ovarian cancer. IMPLICATIONS: Targeting the let-7e-PARP1-DNA repair axis might be an effective strategy for the treatment of chemoresistant ovarian cancer.

Abstract P2-05-05: RAD51 inhibition using CYT-0851, shows anti-cancer activity in cellular models of breast cancer and acts synergistically with PARP inhibitors

Abstract Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. The clinical success of PARP inhibitors has highlighted the potential of targeting these dependencies therapeutically to induce synthetic lethality. We have developed novel RAD51 inhibitors to selectively target cancers that have elevated levels of DNA damage and/or reduced levels of DNA repair. RAD51 is a critical component of the homologous recombination (HR) pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown anti-cancer activity of RAD51 inhibitors in preclinical xenograft mouse models of lymphomas that have elevated rates of DNA mutations due to aberrant expression of Activation Induced Cytidine Deaminase (AID). Breast cancer, due to high rates of genomic instability, may potentially be treated in this fashion. Here we present the characterization of our RAD51 inhibitors in breast cancer. In a murine cell line model of metastatic breast cancer, 4T1, treatment with CYT-0851 resulted in a reduction in homologous recombination activity in a sister chromatid exchange (SCE) assay at a similar rate observed in a human derived cell line (HEK293T) suggesting conservation of mechanism in human and murine models. In the same cell line, treatment with CYT-0851 and a DNA damaging agent, carboplatin, resulted in cellular toxicity above what was observed with CYT-0851, indicating that RAD51 inhibition coupled with genomic instability is cytotoxic in this breast cancer model. PARP inhibitors have been shown to induce genomic instability as measured by an increase in HR activity in SCE assays. We therefore hypothesized that our RAD51 inhibitor could act as a sensitizer to PARP inhibitors. Combinatorial treatment of 4T1 cells with CYT-0851 and olaparib reduced homologous recombination activity in the SCE assay. Additionally, combination therapy led to increased accumulation of gH2AX foci after ionizing radiation, and increased tail moment on neutral Comet assay. Chou-Talalay synergy assay demonstrated synergistic activity between olaparib and CYT-0851 in 4T1. To determine potential drug synergy in human models, a matrix study was performed with CYT-0851 and olaparib in three cell lines of triple negative breast cancer (HCC1937, HCC1143, and BT20). Concentration-dependent synergy was observed in all cell lines tested. These data suggest that CYT-0851 may be active as a mono therapy and a combinatorial therapy with PARP inhibitors in breast cancer. Overall, we conclude that the synergy observed in vitro indicates significant potential for RAD51 inhibition in future cancer treatment strategies and warrants future exploration in vivo. Citation Format: Joey L Guy, Tyler Maclay, Melinda Day, Monika L Burness, Kevin Mills. RAD51 inhibition using CYT-0851, shows anti-cancer activity in cellular models of breast cancer and acts synergistically with PARP inhibitors [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-05-05.

RNF8 promotes efficient DSB repair by inhibiting the pro-apoptotic activity of p53 through regulating the function of Tip60.

ObjectivesRING finger protein 8 (RNF8) is an E3 ligase that plays an essential role in DSB repair. p53 is a well‐established tumour suppressor and cellular gatekeeper of genome stability. This study aimed at investigating the functional correlations between RNF8 and p53 in DSB damage repair.Materials and methodsIn this article, wild‐type, knockout and shRNA‐depleted HCT116 and U2OS cells were stressed, and the roles of RNF8 and p53 were examined. RT‐PCR and Western blot were utilized to investigate the expression of related genes in damaged cells. Cell proliferation, apoptosis and neutral cell comet assays were applied to determine the effects of DSB damage on differently treated cells. DR‐GFP, EJ5‐GFP and LacI‐LacO targeting systems, flow cytometry, mass spectrometry, IP, IF, GST pull‐down assay were used to explore the molecular mechanism of RNF8 and p53 in DSB damage repair.ResultsWe found that RNF8 knockdown increased cellular sensitivity to DSB damage and decreased cell proliferation, which was correlated with high expression of the p53 gene. RNF8 improved the efficiency of DSB repair by inhibiting the pro‐apoptotic function of p53. We also found that RNF8 restrains cell apoptosis by inhibiting over‐activation of ATM and subsequently reducing p53 acetylation at K120 through regulating Tip60.ConclusionsTaken together, these findings suggested that RNF8 promotes efficient DSB repair by inhibiting the pro‐apoptotic activity of p53 through regulating the function of Tip60.

Post-transcriptional regulation of Rad51c by miR-222 contributes cellular transformation.

DNA repair inhibition has been described as an essential event leading to the initiation of carcinogenesis. In a previous study, we observed that the exposure to metal mixture induces changes in the miR-nome of the cells that was correlated with the sub-expression of mRNA involved in processes and diseases associated with metal exposure. From this analysis, one of the miRNAs that shows changes in its expression is miR-222, which is overexpressed in various cancers associated with exposure to metals. In silico studies showed that a possible target for the microRNA-222 could be Rad 51c, a gene involved in the double-stranded DNA repair. We could appreciate that up-regulation of miR-222 reduces the expression both gene and as a protein expression of Rad51c by RT-PCR and immunoblot, respectively. A luciferase assay was performed to validate Rad51c as miR-222 target. Neutral comet assay was performed in order to evaluate DNA double-strand breaks under experimental conditions. Here, we demonstrate that miR-222 up-regulation, directly regulates Rad51c expression negatively, and impairs homologous recombination of double-strand break DNA repair during the initiation stage of cell transformation. This inhibition triggers morphological transformation in a two-stage Balb/c 3T3 cell assay, suggesting that this small RNA acts as an initiator of the carcinogenesis process.

Microsurgical varicocelectomy effect on sperm telomere length, DNA fragmentation and seminal parameters

Varicocele is one of the main causes of male infertility and microsurgical varicocelectomy (MV) seems to be the best procedure for its repair and to reduce testicular oxidative stress (ROS). As ROS causes guanine modifications, we postulated that DNA damage could be more intense in telomeres due to their G-rich nature. We studied the effect of MV on sperm telomere length (TL), single- and double-strand DNA fragmentation (ssSDF and dsSDF) and seminal parameters. Sperm telomeres from 12 fertile donors and 20 varicocele patients before and nine months after MV were labelled using FITC-PNA qFISH (a new method to obtain absolute TL from relative fluorescence intensity using FITC-fluorescent spheres). Both ssSDF and dsSDF were analysed using the alkaline and neutral Comet assays, respectively. The results showed that varicocele and MV had no effect on TL. Seminal parameters, ssSDF and dsSDF of varicocele patients were altered. Although these parameters improved after MV, values did not reach those seen in fertile donors. A good estimation of absolute TL was developed based on FITC-fluorescent spheres. The results showed that TL is not affected by varicocele or surgery. However, MV is able to partially reduce altered seminal parameters, ssSDF and dsSDF values in varicocele patients.

Can Utilization of Sperm DNA Fragmentation (SDA) Tests in Combination with Time Lapse Microscopy Help in Improving ICSI Implantation Rates in Unexplained Recurrent Implantation Failures Utilizing Double Stranded SDA as the Causative Factor for the same

Over 50% of intracytoplasmic sperm injection (ICSI) cycles don’t display implantation. Hence laboratories make their maximum efforts to select the best embryos as far as implantation enhancement is concerned. Further utilization of available technologies like time lapse recording have been made in a large number of artificial reproductive technology (ART) centres. Various studies that utilize embryo kinetics have implicated that time when embryo cleavage may prove to be an important factor that determines the implantation potential of an embryo. With this variety of algorithms mathematic wise have been used to forecast which the best embryos are for transfer. But the efficacy of these might be influenced by multiple confounding factors. Thus work on biomarkers that can forecast good ART warrants newer embryo selection basis. Regarding conventional ICSI, typical standard routine semen analysis involving sperm concentration, motility and morphology does not predict the implantation percentages in an ICSI cycle. Once sperm DNA fragmentation (SDA) methods were inducted they appeared to hold promise in forecasting good ART success. Although certain studies utilizing various techniques like TUNEL. SCSA, SCD proved a relation existed between DNA damage and implantation rates in ICSI but the same was contradicted by others. With this it was thought that bias between evaluation of ejaculate and motile sperm picked up for ICSI, as is known regarding absence of positive association of sperm motility and DNA fragmentation. Thus study by Casanovas et.al., tried to find if there is any correlation of single stranded (ssSDA) and double stranded (dsSDA) sperm DNA damage that might forecast ICSI success and utilizing Neutral Comet Assays along with help of time lapse technology they found that double stranded sperm influenced delay in embryo formation as seen by embryo kinetics and thus interfere with implantation rates. Reproduction of these findings might help in getting a standard for getting best embryos selected in ICSI utilizing SDA and time lapse microscopy

Assessment of avian sperm DNA fragmentation using the sperm chromatin dispersion assay.

Herein we report a simple method for assessing avian sperm DNA fragmentation (SDF) using the sperm chromatin dispersion test (SCDt). The presence of sperm DNA damage was confirmed indirectly by correlating results of the SCDt determined in three bird species with results of a corresponding neutral comet assay (r=0.99; P<0.005). Frozen-thawed spermatozoa of each species were also incubated at 37°C for 5h and the within- and between-species variation of SDF, as an indicator of sperm DNA longevity, examined. The dynamic assessment of SDF using the SCDt revealed species and individual bird (rooster and turkey) differences in sperm DNA longevity.

Cyclin K interacts with β-catenin to induce Cyclin D1 expression and facilitates tumorigenesis and radioresistance in lung cancer.

Rationale: Radioresistance remains the major cause of local relapse and distant metastasis in lung cancer. However, the underlying molecular mechanisms remain poorly defined. This study aimed to investigate the role and regulatory mechanism of Cyclin K in lung cancer radioresistance.Methods: Expression levels of Cyclin K were measured by immunohistochemistry in human lung cancer tissues and adjacent normal lung tissues. Cell growth and proliferation, neutral comet and foci formation assays, G2/M checkpoint and a xenograft mouse model were used for functional analyses. Gene expression was examined by RNA sequencing and quantitative real-time PCR. Protein-protein interaction was assessed by immunoprecipitation and GST pull-down assays.Results: We report that Cyclin K is frequently overexpressed and correlates with poor prognosis in lung cancer patients. Functionally, we demonstrate that Cyclin K depletion results in reduced proliferation, defective G2/M checkpoint and enhanced radiosensitivity in lung cancer. Mechanistically, we reveal that Cyclin K interacts with and promotes the stabilization of β-catenin protein, thereby upregulating the expression of Cyclin D1. More importantly, we show that Cyclin D1 is the major effector that mediates the biological functions of Cyclin K in lung cancer.Conclusions: These findings suggest that Cyclin K positively modulates the β-catenin/Cyclin D1 axis to promote tumorigenesis and radioresistance in lung cancer, indicating that Cyclin K may represent a novel attractive biomarker for lung cancer radiotherapy.

The effect of inhibitors of phosphatidylinositol 3-kinase-related kinases on dibenzo[def,p]chrysene genotoxicity measured by γH2AX levels and neutral comet assay in HepG2 human hepatocellular cancer cells

In the study the modulating effect of inhibition of phosphatidylinositol 3-kinase-related kinases (PIKK): ATM (Ataxia Telangiectasia Mutated), ATR (Ataxia Telangiectasia and Rad3 Related) and DNA-PK (DNA-dependent protein kinase) on genotoxicity of dibenzo[def,p]chrysene (DBC) in HepG2 human hepatocellular cancer cells was investigated. The cytotoxicity of DBC was determined, also in combination with PIKK inhibitors, using the MTT reduction assay. The high cytotoxicity of DBC was observed after 72 h incubation (IC50 = 0.06 μM). The PIKK inhibitors applied at non-cytotoxic concentrations: caffeine (1 mM) and KU55933 (2.5 μM) had no significant influence on the DBC cytotoxicity, however NU7026 (5 μM) caused significant increase in the cell viability by about 25%. The combinations of the inhibitors (double or triple) where NU7026 was present also caused increase in the cell viability (i.e. cytoprotective effect) compared to the effect of DBC. The level of damage to the genetic material (DNA double strand breaks, DSB) was assessed by measuring levels of phosphorylated form of H2A histone (γH2AX) and neutral comet assay. DBC induced DSB in a concentration and time-dependent manner. NU7026 considerably reduced the level of DSB level measured by γH2AX and comet assay.The obtained results confirm that DBC is cytotoxic and causes damage to the genetic material including DSB. The DNA-PK inhibitor NU7026 increases cell viability after exposure to DBC and reduces DNA damage, what indicates an important role of the sensor kinase in mediating the effect.

Biosynthesis of Silver Nanoparticles Using Thymoquinone and Evaluation of Their Radio-Sensitizing Activity

Delivering an optimum radiation dose to the target and preserving the surrounding normal tissues are the main tasks in radiation therapy. Latterly, there is a special interest in formulations of metallic-based nanoparticles to be used as radio-sensitizers. One of these silver nanoparticles (AgNPs) have been used for a wide range of biomedical applications depending on their surface modifications, size, and shape. This study aims to fabricate and characterize green synthesized silver nano-formulations using thymoquinone (TQ), as reducing and capping agent and assessment of their radio-sensitizing effect against the highly aggressive MDA-MB-231 mammary adenocarcinoma. AgNPs were prepared with different sizes by changing the amounts of added TQ. The prepared silver nano-formulations have been characterized using different physical techniques. The radio-sensitization efficacy of prepared AgNPs has been assessed using MTT cytotoxicity assay. Further assessment, neutral comet assay, was carried out to investigate the amount of DNA double-strand breaks which occurred due to AgNPs radio-sensitization. TEM micrograph revealed that as the amount of the added TQ increased from 10, 20, and 40 mg, the average size of prepared silver nanoparticles (AgNPs-20, AgNPs-15, and AgNPs-10) was gradually decreased to be 20 ± 1.5 nm, 15 ± 2 nm, and 10 ± 1.8 nm, respectively. This was confirmed by DLS measurements and UV-Vis spectroscopy. Comet and MTT assay results showed that AgNPs have a size and concentration-dependent radio-sensitization activity, whereas the radiation dose enhancement for small-sized nanoparticles (AgNPs-10) was greater than that of larger sized (AgNPs-20 and AgNPs-15). Therefore, thymoquinone capping silver nanoparticles represent a promising engineered nano-formulation for enhancing cancer radiosensitivity.

Targeting DNA Damage and Repair in Acute Myeloid Leukemia Carrying Internal Tandem Duplication of Fms-like Tyrosine Kinase 3 (FLT3-ITD) - a Mechanistic Study

Objective Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) is one of the most common mutations in acute myeloid leukemia (AML), occurring in nearly 30% of cases. FLT3-ITD involves in-frame duplication of 3-400 base-pairs at the juxta-membrane domain, resulting in ligand-independent activation of FLT3 signaling. Downstream effectors include activation of ERK/STAT5 via SRC kinase, activation of PI3K/AKT, phosphorylation of FOXO3A, down-regulation of equilibrative nucleoside transporter 1 (ENT1) for cytarabine, and induction of reactive oxygen species (ROS) that may lead to increased DNA damage and defective repair. The present study investigated if the latter can be effectively targeted for the treatment of this AML subtype. Methods Primary samples from patients with FLT3-ITD AML, human cell line carrying FLT3-ITD (MOLM-13) as well as mouse B-lymphoid Ba/F3 cells transduced with human FLT3-ITD were used in this study. Traffic Light Reporter (TLR) assay was used to measure fidelity of double-strand breaks (DSB) repair, either via error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Percentage of repair events by HR or NHEJ were quantified by flow cytometry. DNA DSB were examined by γ-H2AX foci staining using confocal microscopy. Single-cell DSB were quantified by neutral comet assay and analysed by OpenComet software. The tail moment was calculated as the length of comet tail multiplied by the tail DNA %. MOLM-13 was transplanted into NOD/SCID/IL2Rg-/- (NSG) mice by tail vein injection. Treatment comprised cytarabine (25mg/kg, i.p., day 1-5) and doxorubicin (1.5mg/kg, i.v., day 1-3), with or without olaparib (25mg/kg, i.p., day 1-5). Comparisons between groups of numerical data were evaluated using Student's t-test. P-values less than 0.05 were considered statistically significant. Results To investigate the link between FLT3-ITD AML and DNA damage response (DDR), expression of critical DDR genes in primary AML samples was examined by real-time quantitative PCR. The panel of genes included apical kinase ATM, ATR and DNA-PKcs; DNA damage mediators BRCA1,BRCA2 and PARP1; downstream response kinase CHEK1 and CHEK2 and effectors TP53. Among them, BRCA2 was significantly down-regulated in FLT3-ITD AML(Wild-type FLT3=18 samples; FLT3-ITD=13 samples; Average dCt 9.7 in WT vs. 10.6 in ITD; p&lt;0.05). Down-regulation of BRCA2 in FLT3-ITD AML was further validated in a microarray database GSE15434 from a multicenter study investigating gene expression profiles of normal karyotype AML( FLT3-WT=148; FL3-ITD=86; ; log2 gene expression 5.61 in WT vs 5.45 in ITD; p&lt;0.001). As BRCA2 is an important protein mediating HR, a DSB DNA repair TLR assay was performed. HR was significantly down-regulated in Ba/F3-FLT3-ITD as compared with parental Ba/F3 line by flow cytometry(1.21% HR repair in control vs. 0.44% in ITD; p&lt;0.05) while NHEJ was unaffected (1.90% NHEJ repair in control vs. 2.38% in ITD; p=0.50). Down-regulation of BRCA2 expression and defective HR in FLT3-ITD AML was reminiscent of BRCA mutant breast and ovarian cancers. Therefore, the effects of poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib were examined. Ba/F3-FLT3-ITD were more sensitive to olaparib than parental line. Olaparib inhibited base excision repair and increased DSB as indicated by increased number of γ-H2AX foci and increased tail moment in Ba/F3-FLT3-ITD cells when compared to parental line. Olaparib-induced DSB in Ba/F3-FLT3-ITD cells was mainly repaired by NHEJ as shown by colocalization of γ-H2AX foci with 53BP1. Combination of chemotherapy (cytarabine and doxorubicin) and olaparib synergistically reduced leukemic cell growth of MOLM-13 in NSG murine xenograft model. Conclusion Results from the present study supported the use of PARPi in the treatment of FLT3-ITD AML. Its therapeutic benefits in combination with chemotherapy would have to be further evaluated. Acknowledgements: Health and Medical Research Fund (Project number: 04152326) and Li Shu Fan Medical Foundation, LKS Faculty of Medicine, University of Hong Kong. Disclosures No relevant conflicts of interest to declare.

Oxidative DNA damage is concurrently repaired by base excision repair (BER) and apyrimidinic endonuclease 1 (APE1)-initiated nonhomologous end joining (NHEJ) in cortical neurons.

AimsAccumulating studies have suggested that base excision repair (BER) is the major repair pathway of oxidative DNA damage in neurons, and neurons are deficient in other DNA repair pathways, including nucleotide excision repair and homologous recombination repair. However, some studies have demonstrated that neurons could efficiently repair glutamate‐ and menadione‐induced double‐strand breaks (DSBs), suggesting that the DSB repair mechanisms might be implicated in neuronal health. In this study, we hypothesized that BER and nonhomologous end joining (NHEJ) work together to repair oxidative DNA damage in neurons.MethodsImmunohistochemistry and confocal microscopy were employed to examine the colocalization of apyrimidinic endonuclease 1 (APE1), histone variant 2AX (γH2AX) and phosphorylated p53‐binding protein (53BP1). APE1 inhibitor and shRNA were respectively applied to suppress APE1 activity and protein expression to determine the correlation of APE1 and DSB formation. The neutral comet assay was used to determine and quantitate the formation of DSB.ResultsBoth γH2AX and 53BP1 were upregulated and colocalized with APE1 in the nuclei of rat cortical neurons subjected to menadione‐induced oxidative insults. Phospho53BP1 foci were efficiently abolished, but γH2AX foci persisted following the suppression of APE1 activity. Comet assays demonstrated that the inhibition of APE1 decreased the DSB formation.ConclusionsOur results indicate that APE1 can engage the NHEJ mechanism in the repair of oxidative DNA damage in neurons. These findings provide insights into the mechanisms underlying the efficient repair of oxidative DNA damage in neurons despite the high oxidative burden.

Inhibiting BRAF Oncogene–Mediated Radioresistance Effectively Radiosensitizes BRAFV600E-Mutant Thyroid Cancer Cells by Constraining DNA Double-Strand Break Repair

Activating BRAF mutations, most commonly BRAFV600E, are a major oncogenic driver of many cancers. We explored whether BRAFV600E promotes radiation resistance and whether selectively targeting BRAFV600E with a BRAF inhibitor (vemurafenib, BRAFi) sensitizes BRAFV600E thyroid cancer cells to radiotherapy. Immunoblotting, neutral comet, immunocytochemistry, functional reporter, and clonogenic assays were used to analyze the outcome and molecular characteristics following radiotherapy with or without BRAFV600E or vemurafenib in thyroid cancer cells. BRAFV600E thyroid cancer cell lines were associated with resistance to ionizing radiation (IR), and expression of BRAFV600E into wild-type BRAF thyroid cancer cells led to IR resistance. BRAFi inhibited ERK signaling in BRAFV600E mutants, but not BRAF wild-type thyroid cancer cell lines. BRAFi selectively radiosensitized and delayed resolution of IR-induced γH2AX nuclear foci in BRAFV600E cells. Moreover, BRAFi impaired global DNA repair and altered the resolution of 53BP1 and RAD51 nuclear foci in BRAFV600E cells following IR. BRAFV600E mutants displayed enhanced nonhomologous end-joining (NHEJ) repair activity, which was abolished by BRAFi. Intriguingly, BRAFV600E mutation led to upregulation of XLF, a component of NHEJ, which was prevented by BRAFi. Importantly, BRAFi in combination with radiotherapy resulted in marked and sustained tumor regression of BRAFV600E thyroid tumor xenografts. BRAFV600E mutation promotes NHEJ activity leading to radioresistance and BRAFi selectively radiosensitizes BRAFV600E thyroid cancer cells through inhibiting NHEJ. Our findings suggest that combining BRAFi and radiation may improve the therapeutic outcome of patients with BRAFV600E-mutant thyroid cancer.

Cisplatin and Vinorelbine -Mediated Electrochemotherapeutic Approach Against Multidrug Resistant Small Cell Lung Cancer (H69AR) In Vitro.

Small cell lung cancer (SCLC) originates from neuroendocrine branchial cells (15-20%). It is regarded as distinct from other lung cancers due to its biological and clinical features. In most cases of SCLC, surgery or radiotherapy alone is not an effective cure. The aim of our study was to examine the cytotoxic effects of chemotherapy supported by electroporation (EP) on a resistant SCLC model, in vitro. The multidrug resistant small lung cell line H69AR was used to evaluate the cytotoxic effects of cisplatin (CPPD) and vinorelbine (Navirel®; NAV) at lower doses when used with EP. Cells were treated with different concentrations of CPPD and NAV, alone or in combination with the following EP parameters: 400-1200 V/cm, 8 pulses of 100 μs duration, at 1Hz. The cell viability was estimated by MTT assay after 24 and 48 h. Apoptotic cells were detected by neutral comet assay and immunofluorescence assay with PARP-6. CPPD and NAV alone showed a dose-dependent effect on cell viability. Cytostatic drugs combined with EP revealed increased anticancer activity. Lower doses of CPPD or NAV delivered by EP were as effective as higher doses of these drugs without EP. The electrochemotherapeutic protocols increased the number of apoptotic cells and increased immunoreactivity of PARP-6. Our results indicated higher sensitivity of H69AR cells to NAV supported by EP. In SCLC cells, an increased anticancer activity was potentiated by exposure of cells to high intensity electric pulses and low drug doses. It is suggested that this method could be effectively applied in the treatment of lung cancer.

PON1 increases cellular DNA damage by lactone substrates

Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme that by hydrolysing exogenous and endogenous substrates can provide protection against substrate induced toxicity. To investigate the extent to which PON1 provides protection against lactone induced DNA damage, DNA damage was measured in HepG2 cells using the neutral Comet assay following lactone treatment in the presence and absence of exogenous recombinant PON1 (rPON1). Low dose lactones (10 mM) caused little or no damage while high doses (100 mM) induced DNA damage in the following order of potency: α-angelica lactone > γ-butyrolactone ~ γ-hexalactone > γ-heptalactone ~ γ-octaclactone ~ γ-furanone ~ γ-valerolactone > γ-decalactone. Co-incubation of 100 mM lactone with rPON1, resulted in almost all cells showing extensive DNA damage, particularly with those lactones that decreased rPON1 activity by > 25%. In contrast, with the lactones that are poor rPON1 subtrates (γ-decalactone and γ-furanone), rPON1 did not increase DNA damage. DNA damage induced by a 1 h co-treatment with 10 mM α-angelica lactone and rPON1 was reduced when cells when incubated for a further 4 h in fresh medium suggesting break formation was due to induced DNA damage rather than apoptosis. Preincubation (1–6 h) of α-angelica lactone with rPON1 in the absence of cells, decreased cellular DNA damage by around 40% in comparison to cells treated without preincubation. These results suggest that in addition to its well-recognised detoxification effects, PON1 can increase genotoxicity potentially by hydrolysing certain lactones to reactive intermediates that increase DNA damage via the formation of DNA adducts.

The influence of kinesin light chain-2 on the radiosensitivity of non-small cell lung cancer cells and the underlying mechanisms.

e14512 Background: Our previous study found upregulated kinesin light chain-2 (KLC2) in non-small cell lung cancer (NSCLC) cell lines and tissues, which is associated with a poor prognosis. KLC2 promotes the migration ability of lung cancer cells. However, the influence of KLC2 on radiosensitivity of NSCLC has not yet been reported. Methods: Sensitivity of lung cancer cells to radiation was analyzed by colony formation, γH2AX immunofluorescent staining assay and neutral comet assay in vitro and a xenograft tumor model in vivo. Gene set enrichment analysis (GSEA), qRT-PCR and western blot assay were performed to predict and validate the potential target genes of KLC2. Results: We found that down-regulation of KLC2 could significantly improve the radio-sensitivity of lung cancer cells, while the overexpression of KLC2 had the opposite effect. GSEA identified p53 signal pathway as differentially enriched with high KLC2 expression, which was validated by western blot assay. Using data from TCGA, we found a positive correlation between the mRNA level of KLC2 and that of HuR. The expression of KLC2 mRNA and protein in lung cancer cells was significantly reduced when downregulating HuR. Interestingly, the overexpression of KLC2 could also significantly decrease the expression of HuR. Conclusions: The above results indicated that HuR could upregulate the expression of KLC2, which might decrease the phosphorylation of p53 and thereby weakened the radio-sensitivity of NSCLC cells. KLC2 could also positively regulate the expression of HuR as a positive feedback. Support: 81572279, 2016J004, LC2016PY016, 2018CR033

Stress hormones induce DNA damage in ovarian cancer cells

Activation of the sympathetic nervous system (SNS) is associated with cancer progression. Recent data suggest that adrenergic signaling alters the genetic integrity of cancer cells by damaging the DNA and/or influencing the DNA‐maintenance machinery composed of the DNA Damage Response Pathway and DNA Repair Pathways. Consequently, accumulation of genomic alterations can lead to genomic instability and the acquisition of other cancer hallmarks that drive tumor progression. Still, the exact mechanisms of how sustained adrenergic signaling influences genomic stability is an area that needs to be further studied. We hypothesize that increased adrenergic stimuli will induce significant levels of DNA damage. To assess the extent of DNA damage after catecholamine exposure, we determined p‐H2AX (sensitive marker for double‐stranded DNA damage) expression by western blot and immunofluorescence analyses in SKOV3 and other high‐grade serous ovarian cancer cell lines. Neutral Comet Assays were performed to assess double‐stranded DNA damage after catecholamine exposure. SKOV3 cells that were exposed to epinephrine (Epi) and norepinephrine (Ne) at 1h, 3h, 6h, 12h, and 24h displayed increased expression of p‐H2AX. Moreover, immunofluorescence analyses for p‐H2AX in SKOV3 and OV90 showed increased foci formation (reflecting 1:1 foci/double‐strand break) when compared to the control group. Lastly, Neutral Comet Assay analyses in catecholamine‐treated SKOV3 and COV362 cells displayed higher levels of double‐stranded DNA breaks in comparison to untreated cells. Lastly, to determine if DNA damage is due to adrenergic stimuli, cells were pre‐treated with propranolol (non‐specific β‐blocker) and results showed a significant reduction in the levels of DNA damage. These results suggest that catecholamines can induce double‐stranded DNA damage in ovarian cancer cells.Support or Funding InformationNIMHHD (G12MD007579), NIGMS‐RISE R25GM082406, NCI (U54CA163071 and U54CA163068), U54CA163071‐06, and Ponce Research Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of 177Lu-iPSMA on viability and DNA damage of human glioma cells subjected to hypoxia-mimetic conditions

The therapeutic potential of 177Lu-iPSMA on hypoxic cancer cells has not been yet demonstrated. The aim of this work was to evaluate the radiation dose effect of 177Lu-iPSMA on viability and DNA damage in U87MG human glioma cells subjected to hypoxia-mimetic conditions. U87MG cells treated with 177Lu-iPSMA were incubated with CoCl2 in order to induce hypoxia-mimetic conditions. The cytotoxic and genotoxic effect was evaluated with an in vitro viability test and a neutral comet assay. 177Lu-iPSMA decreased the cell viability and induced DNA double strand breaks in U87MG human glioma cells under hypoxia-mimetic conditions. 177Lu-iPSMA produced the maximum effect at 48 h, suggesting that this radiopharmaceutical could be used as a strategy for the treatment of human glioma hypoxic cells.