Formation Of Strand Breaks Research Articles

Abstract P2-05-06: Nuclear STING localization induces chemoresistance in breast cancer

Abstract Efficient DNA damage repair (DDR) is crucial for a cell as aberrant repair can create deleterious mutations and genomic instability while unrepaired damage leads to cell apoptosis. Cells with altered DDR are more inclined to develop a variety of diseases, including cancers Cancer therapies including radiotherapy and chemotherapy rely on double strand break (DBS) formation to drive the killing of rapidly cycling tumor cells. As efficient DNA repair pathways can enable tumor cells to survive treatment-induced DNA damage, the identification of new actors participating in efficient DDR will help design alternative therapeutic approaches. Here we report that the well-known DNA sensor of innate immunity Stimulator of Interferon Genes (STING) is directly involved in the modulation of DSB repair efficiency in breast cancer cells. STING partly resides at the inner membrane of the nucleus where it interacts with core proteins of DNA-damage complexes and promotes its assembly with chromatin. STING silencing decreases DNA repair and cell viability, while STING overexpression protects cancer cells from genotoxic treatment. STING involvement in DNA repair is independent of the canonical STING-triggered pro-inflammatory response. In line with this observation, while overall STING expression level is positively correlated with favorable outcome in breast cancer patients, high STING expression in breast and ovarian cancer patients treated with adjuvant chemotherapy is associated with poor prognosis. This newly-identified function of STING elucidates a cell autonomous tumor-promoting mechanism complementary and independent from its well-documented role in tumor immunosurveillance. Citation Format: Laura Cheradame, Chiara Guerrera, Jean-Gabriel Judde, Vincent Goffin, Stefano Cairo. Nuclear STING localization induces chemoresistance in breast cancer [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 P2-05-06.

Cellular Reference Materials for DNA Damage Using Electrochemical Oxidation.

Reference materials are needed to quantify the level of DNA damage in cells, to assess sources of measurement variability and to compare results from different laboratories. The comet assay (single cell gel electrophoresis) is a widely used method to determine DNA damage in the form of strand breaks. Here we examine the use of electrochemical oxidation to produce DNA damage in cultured mammalian cells and quantify its percentage using the comet assay. Chinese hamster ovary (CHO) cells were grown on an indium tin oxide electrode surface and exposed 12 h to electrochemical potentials ranging from 0.5 V to 1.5 V (vs Ag/AgCl). The resulting cells were harvested and analyzed by comet and a cell viability assay. We observed a linear increase in the percentage (DNA in tail) of strand breaks along with a loss of cell viability with increasing oxidation potential value. The results indicate that electrochemically induced DNA damage can be produced in mammalian cells under well-controlled conditions and could be considered in making a cellular reference material for the comet assay.

Aluminium Induced DNA-damage and Oxidative Stress in Cultures of the Marine Sponge Hymeniacidon perlevis

Aluminium is the most abundant element in the earth crust, and has no known biological function. However, it is an established neurotoxicant in its trivalent oxidation state, with exposure resulting in neurodegenerative diseases like Parkinson’s disease and presenile dementia. Although, the potential genotoxic and carcinogenic effects of aluminium are established in mammalian and other model system, there is however very limited information on aluminium genotoxicity in aquatic invertebrates. Mechanism of aluminium toxicity is also largely unclear. With a concentration range between 0.001– 0.05mg/L in near neutral pH water, and up to 0.5-1mg/L in an acidic water , aluminium poses a potential threat to the marine ecosystem, however it is poorly studied. This study, therefore presents for the first time, aluminium-induced DNA damage using the comet assay and reactive oxygen Species (ROS) formation using 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCF-DA) assay as biomarkers of genotoxicity and oxidative stress in the inter-tidal marine sponge Hymeniacidon perlevis, respectively. H. perlevis is widely distributed in the British Isles, Mediterranean and the Arctic sea and has been reported as a model for environmental biomonitoring in aquatic ecosystem and as a suitable alternative to bivalves. In this study, cryopreserved single sponge cells of H. perlevis were cultured as viable aggregates and were thereafter treated with 0.1, 0.2, 0.3 and 0.4mg/L aluminium chloride (AlCl3) for 12 hours. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Our results showed that non-cytotoxic concentrations of AlCl3 caused a statistically significant concentration-dependent increase in the level of DNA-strand break and reactive oxygen species formation single sponge cells of H. perlevis. There was also a statistically significant positive linear correlation between aluminium-induced DNA strand break and ROS formation suggesting the involvement of ROS in the causative mechanism of the aluminium induced DNA-strand breaks observed.

SPDR-01 INHIBITION OF HOMOLOGOUS RECOMBINATION, PARP INHIBITOR, OR DIANHYDROGALACTITOL OVERCOMES TEMOZOLOMIDE-RESISTANCE IN GLIOMA CELLS

Glioblastoma is one of the most aggressive tumors, with 5-year survival rates of less than 10%. The standard therapy for glioblastomas is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide (TMZ). The poor prognosis is partially contributed to the acquisition of resistance to TMZ and intratumoral heterogeneity. The mechanisms of resistance to TMZ are various due to tumor heterogeneity. TMZ is a DNA-methylating agent, delivering a methyl group to DNA (O6-guanine, N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine, mispairs with thymine, leading to futile DNA mismatch repair (MMR), formation of double strand breaks (DSBs) and eventual cell death, when O6-methylguanine DNA methyltransferase (MGMT) is absent. N7-methylguanine and N3-methyladenine are repaired by base excision repair (BER). The object of the study was to reveal the mechanisms of resistance to TMZ and to find the way to overcome the resistance in glioma. Several clones of TMZ-resistant U251 or U87 were obtained and analyzed. Increased homologous recombination (HR) and deficiency of MMR system, not MGMT were revealed to be contributed to the resistance to TMZ. Inhibition of HR resensitized cells with high HR to TMZ, but it could not resensitize cells with deficient MMR. For the cells with deficient MMR, Inhibition of BER by PARP inhibitor was revealed to potentiate the TMZ-induced cytotoxicity. PARP inhibitors also potentiate the cytotoxicity of TMZ to cells with expressed MGMT. Dianhydrogalactitol (DAG) is a bifunctional DNA-targeting agent, forming N7 alkylguanine and inter-strand DNA crosslinks. DAG reduced the proliferation of cells independent of MGMT and MMR, inducing DNA DSBs, G2/M arrest, and apoptosis in TMZ-resistant glioma cells. Inhibition of chk1, or HR could enhance the cytotoxicity of DAG, increasing apoptosis cells. By selecting the appropriate treatments to the types of resistant mechanisms, these new treatments have the potential to improve the prognosis of glioblastoma.

Shining light on the response to repair intermediates in DNA of living cells

Fluorescently-tagged repair proteins have been widely used to probe recruitment to micro-irradiation-induced nuclear DNA damage in living cells. Here, we quantify APE1 dynamics after micro-irradiation. Markers of DNA damage are characterized and UV-A laser micro-irradiation energy conditions are selected for formation of oxidatively-induced DNA base damage and single strand breaks, but without detectable double strand breaks. Increased energy of laser micro-irradiation, compared with that used previously in our work, enables study of APE1 dynamics at the lesion site. APE1 shows rapid transient kinetics, with recruitment half-time of less than 1 s and dissociation half-time of less than 15 s. In cells co-transfected with APE1 and PARP1, the recruitment half-time of PARP1 was slower than that of APE1, indicating APE1 is a rapid responder to the damage site. While recruitment of APE1 is unchanged in the presence of co-transfected PARP1, APE1 dissociation is 3-fold slower, revealing PARP1 involvement in APE1 dynamics. Further, we find that APE1 dissociation kinetics are strongly modified in the absence of DNA polymerase β (pol β). After unchanged recruitment to the damage site, dissociation of APE1 became undetectable. This indicates a necessary role for pol β in APE1 release after its recruitment to the damage site. These observations represent an advance in our understanding of in vivo dynamics of base excision repair factors APE1, PARP1 and pol β.

DRES-03. PARP INHIBITOR, INHIBITION OF HOMOLOGOUS RECOMBINATION, OR DIANHYDRODULCITOL CAN OVERCOME TEMOZOLOMIDE-RESISTANCE IN GLIOMA CELLS

Abstract Glioblastoma is one of the most aggressive tumors in the central nervous system tumors, with 5-year survival rates of less than 10%. The standard therapy for glioblastomas is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide (TMZ). The acquisition of resistance to TMZ and the heterogeneity of the tumor are considered to be associated with poor prognosis. TMZ is a DNA-methylating agent, delivering a methyl group to DNA (O6-guanine, N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine, mispairs with thymine, leading to futile DNA mismatch repair (MMR), formation of double strand breaks and eventual cell death, when O6-methylguanine DNA methyltransferase (MGMT) is absent. N7-methylguanine and N3-methyladenine are repaired by base excision repair (BER). The object of the study was to reveal the mechanisms of resistance to TMZ and to find the way to overcome the resistance in glioma. Several clones of TMZ-resistant U251 were obtained and analyzed. A clone showed high homologous recombination (HR), and inhibition of HR resensitized the clone to TMZ. MMR system was suppressed in the other clones. Inhibition of BER by PARP inhibitor resensitized them to TMZ. PARP inhibitor enhanced the TMZ-induced cytotoxicity in glioma cells with high expression of MGMT. Dianhydrodulcitol, alkylating agent, suppressed the proliferation in U251 and even U251-derived resistant clones. These effects were shown in U87MG-dereived resistant clones and MGMT-proficient cell lines. Moreover, inhibition of HR enhanced the cytotoxicity of dianhydrodulcitol. To select the appropriate treatment according to the mechanisms of resistance to TMZ can overcome the resistance in glioma cells.

Homozygous mutations in REC114 cause female infertility characterised by multiple pronuclei formation and early embryonic arrest

BackgroundAbnormal pronuclear formation during fertilisation and subsequent early embryonic arrest results in female infertility. In recent years, with the prevalence of assisted reproductive technology, a few genes have been identified...

Interplay between Caspase 9 and X-linked Inhibitor of Apoptosis Protein (XIAP) in the oocyte elimination during fetal mouse development

Mammalian female fertility is limited by the number and quality of oocytes in the ovarian reserve. The number of oocytes is finite since all germ cells cease proliferation to become oocytes in fetal life. Moreover, 70–80% of the initial oocyte population is eliminated during fetal and neonatal development, restricting the ovarian reserve. Why so many oocytes are lost during normal development remains an enigma. In Meiotic Prophase I (MPI), oocytes go through homologous chromosome synapsis and recombination, dependent on formation and subsequent repair of DNA double strand breaks (DSBs). The oocytes that have failed in DSB repair or synapsis get eliminated mainly in neonatal ovaries. However, a large oocyte population is eliminated before birth, and the cause or mechanism of this early oocyte loss is not well understood. In the current paper, we show that the oocyte loss in fetal ovaries was prevented by a deficiency of Caspase 9 (CASP9), which is the hub of the mitochondrial apoptotic pathway. Furthermore, CASP9 and its downstream effector Caspase 3 were counteracted by endogenous X-linked Inhibitor of Apoptosis (XIAP) to regulate the oocyte population; while XIAP overexpression mimicked CASP9 deficiency, XIAP deficiency accelerated oocyte loss. In the CASP9 deficiency, more oocytes were accumulated at the pachytene stage with multiple γH2AFX foci and high LINE1 expression levels, but with normal levels of synapsis and overall DSB repair. We conclude that the oocytes with LINE1 overexpression were preferentially eliminated by CASP9-dependent apoptosis in balance with XIAP during fetal ovarian development. When such oocytes were retained, however, they get eliminated by a CASP9-independent mechanism during neonatal development. Thus, the oocyte is equipped with multiple surveillance mechanisms during MPI progression to safe-guard the quality of oocytes in the ovarian reserve.

1727P - Expression analysis of NHEJ and HR genes in Ewing sarcomas: Indications of DSB repair dysfunction

BackgroundEwing sarcomas (ES) are malignant mesenchymal neoplasms composed of blue small round cells. Ewing sarcomas genetically are characterized mainly by the translocation t(11;22)(q24;q12), which creates EWS/FLI1 fusion gene. These translocations require Double Strand Break (DSB) formation and subsequent dysfunctional repair. BRCAness and HR deficiency have been reported in ES and clinical trials targeting DSB repair are currently ongoing. MethodsWe have studied the expression of Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) genes in 32 cases of Ewing sarcoma. Baseline clinicopathological characteristics were recorded from the medical files. The expression of the following genes XRCC4, XRCC5, XRCC6, Pol λ, Pol m, Lig4, RAD51, RAD52, RAD54, BRCA1, BRCA2, FRANCC, FRANCD, DNMT1 and BRIT1 was analyzed with Real-Time PCR. Samples from healthy blood donors were used as controls. Statistical analysis was performed using SPSS Anova. ResultsNine genes out of the 15 studied showed statistically significant results. XRCC5, XRCC6, Polm, lig4 from the NHEJ DNA repair mechanism and RAD51, RAD52, RAD54, BRCA2 and FRANCD from the HR DNA repair mechanism were upregulated. Interestingly, several parts of both DSB repair mechanisms seem to be dysfunctional highlighting the involvement of NHEJ and HR in the oncogenesis of ES. ConclusionsGenes involved both to NHEJ and HR show statistically important differences in their expression in Ewing sarcoma tumor samples. DSB repair mechanisms seem to be upregulated in a manner that supports the reported articles implying that it could be a potentially important target for new therapeutic approach to these lethal tumors. Legal entity responsible for the studyThe authors. FundingHellenic Society of Medical Oncology. DisclosureAll authors have declared no conflicts of interest.

Conserved HORMA domain-containing protein Hop1 stabilizes interaction between proteins of meiotic DNA break hotspots and chromosome axis.

HORMA domain-containing proteins such as Hop1 play crucial regulatory roles in various chromosomal functions. Here, we investigated roles of the fission yeast Hop1 in the formation of recombination-initiating meiotic DNA double strand breaks (DSBs). Meiotic DSB formation in fission yeast relies on multiple protein-protein interactions such as the one between the chromosome axial protein Rec10 and the DSB-forming complex subunit Rec15. Chromatin immunoprecipitation sequencing demonstrated that Hop1 is colocalized with both Rec10 and Rec15, and we observed physical interactions of Hop1 to Rec15 and Rec10. These results suggest that Hop1 promotes DSB formation by interacting with both axis components and the DSB-forming complex. We also show that Hop1 binding to DSB hotspots requires Rec15 and Rec10, while Hop1 axis binding requires Rec10 only, suggesting that Hop1 is recruited to the axis via Rec10, and to hotspots by hotspot-bound Rec15. Furthermore, we introduced separation-of-function Rec10 mutations, deficient for interaction with either Rec15 or Hop1. These single mutations and hop1Δ conferred only partial defects in meiotic recombination, while the combining the Rec15-binding-deficient rec10 mutation with hop1Δ synergistically reduced meiotic recombination, at least at a model hotspot. Taken together, Hop1 likely functions as a stabilizer for Rec15–Rec10 interaction to promote DSB formation.

Abstract 3823: Inhibition of homologous recombination, PARP inhibitor, or dianhydrodulcitol overcomes temozolomide-resistance in glioma cells

Abstract Glioblastoma is one of the most aggressive tumors in the central nervous system tumors. The standard therapy for glioblastomas is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide (TMZ). The acquisition of resistance to TMZ is big problem. TMZ is a DNA-methylating agent, delivering a methyl group to DNA (O6-guanine, N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine, mispairs with thymine, leading to futile DNA mismatch repair (MMR), formation of double strand breaks and eventual cell death, when O6-methylguanine DNA methyltransferase (MGMT) is absent. N7-methylguanine and N3-methyladenine are repaired by base excision repair. The object of the study was to reveal the mechanisms of resistance to TMZ and to find the way to overcome the resistance in glioma cells. Several clones of TMZ-resistant U251 were obtained and analyzed. #3 clone showed G2 arrest after TMZ exposure and this arrest was abrogated sooner compared to parental U251. TMZ did not induce G2 arrest in #8 clone. The expression of MGMT was not found in U251 parental cells, #3 cells nor #8 cells. The ability of homologous recombination (HR) was increased in #3 clone, and suppression of HR resensitized #3 clone to TMZ. The protein levels of MSH6, which was one of the main proteins associated with MMR, was reduced in #8 clone. Inhibition of base excision repair by PARP inhibitor resensitized #8 clone to TMZ. Dianhydrodulcitol, alkylating agent, suppressed the proliferation in U251 and even U251-derived resistant clones. These effects were shown in U87MG-dereived resistant clones and MGMT-proficient cell lines. To select the way according to the mechanisms of resistance to TMZ could overcome the resistance in glioma cells. Citation Format: SHIGEO OHBA, Yuichi Hirose. Inhibition of homologous recombination, PARP inhibitor, or dianhydrodulcitol overcomes temozolomide-resistance in glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3823.

DNA damage tolerance in stem cells, ageing, mutagenesis, disease and cancer therapy.

The DNA damage response network guards the stability of the genome from a plethora of exogenous and endogenous insults. An essential feature of the DNA damage response network is its capacity to tolerate DNA damage and structural impediments during DNA synthesis. This capacity, referred to as DNA damage tolerance (DDT), contributes to replication fork progression and stability in the presence of blocking structures or DNA lesions. Defective DDT can lead to a prolonged fork arrest and eventually cumulate in a fork collapse that involves the formation of DNA double strand breaks. Four principal modes of DDT have been distinguished: translesion synthesis, fork reversal, template switching and repriming. All DDT modes warrant continuation of replication through bypassing the fork stalling impediment or repriming downstream of the impediment in combination with filling of the single-stranded DNA gaps. In this way, DDT prevents secondary DNA damage and critically contributes to genome stability and cellular fitness. DDT plays a key role in mutagenesis, stem cell maintenance, ageing and the prevention of cancer. This review provides an overview of the role of DDT in these aspects.

DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds in embryo-fetal chicken livers

DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds were assessed in embryo-fetal chicken livers. Formation of DNA adducts and strand breaks were measured using the nucleotide 32P-postlabelling (NPL) and comet assays, respectively. Unsubstituted monocyclic benzene, polycyclic fused ring compound naphthalene, covalently connected polycyclic ring compound biphenyl, and heterocyclic ring compound fluorene did not produce DNA damage. Amino-substituted monocyclic compounds, aniline and p-phenylenediamine, as well as polycyclic 1-naphthylamine were also negative. In contrast, carcinogenic monocyclic methyl-substituted anilines: o-toluidine, 2,6-xylidine, 3,4-dimethylaniline, 4-chloro-o-toluidine; 2 methoxy-substituted methylaniline: p-cresidine; 2,4 and 2,6 diamino- or dinitro- substituted toluenes all produced DNA damage. Genotoxic polycyclic amino-substituted 2-naphthylamine, 4-aminobiphenyl, benzidine, methyl-substituted 3,2′-dimethyl-4-aminobiphenyl and 4-dimethylaminoazobenzene as well as amino- and nitro- fluorenes substituted at the 1 or 2 positions also were positive in at least one of the assays. Overall, the DNA damaging activity of cyclic compounds in embryo-fetal chicken livers reflected the type and position of the substitution on the aromatic ring. Additionally, substituted polycyclic compounds exhibited higher DNA-damaging potency compared to monocyclic chemicals. These results are congruent with in vivo findings in other species, establishing chicken eggs as a reliable system for structure-activity assessment of members of groups of related chemicals.

Genotoxic activity of bisphenol A and its analogues bisphenol S, bisphenol F and bisphenol AF and their mixtures in human hepatocellular carcinoma (HepG2) cells

The use of bisphenol A (BPA) in manufacturing of plastics is being gradually replaced by presumably safer analogues such as bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF). Despite their widespread occurrence in the environment, there is a knowledge gap in their toxicological profiles. We investigated cytotoxic/genotoxic effects as well as changes in the expression of selected genes involved in the xenobiotic metabolism, response to oxidative stress and DNA damage upon exposure to BPs and their mixtures in human hepatocellular carcinoma HepG2 cells.BPS and BPF slightly decreased the viability of HepG2 cells, while BPAF was the most cytotoxic compound tested. BPA, BPF and BPAF induced the formation of DNA double strand breaks determined with γH2AX assay, while BPS was inactive (5–20 μg/mL). All four BPs up-regulated the expression of CYP1A1 and UGT1A1, while BPS up-regulated and BPAF down-regulated also the expression of GST1A. Only BPA up-regulated oxidative stress responsive gene GCLC, while BPAF up-regulated the expression of CDKN1A and GADD45a.At concentrations relevant for human exposure (ng/mL range) BPA and its analogues as individual compounds and in mixtures did not exert genotoxic activity, whereas BPA and BPAF as well as the mixtures up-regulated the expressions of CYP1A1 and UGT1A1.

Propolis Extracts Inhibit UV-Induced Photodamage in Human Experimental In Vitro Skin Models.

The aim of this study was to assess the antioxidant, photoprotective, and antiaging effects of Greek propolis. Propolis was subjected to n-heptane or methanol extraction. Total phenolic/flavonoid content and antioxidant potential were determined in the extracts. Promising extracts were evaluated for their cytoprotective properties using human immortalized keratinocyte (HaCaT) or reconstituted human skin tissue following exposure to UVB. Assessment of cytotoxicity, DNA damage, oxidative status, and gene/protein expression levels of various matrix metalloproteinases (MMPs) were performed. The propolis methanolic fractions exhibited higher total phenolic and flavonoid contents and significant in vitro antioxidant activity. Incubation of HaCaT cells with certain methanolic extracts significantly decreased the formation of DNA strand breaks following exposure to UVB and attenuated UVB-induced decrease in cell viability. The extracts had no remarkable effect on the total antioxidant status, but significantly lowered total protein carbonyl content used as a marker for protein oxidation in HaCaT cells. MMP-1, -3, -7, and -9, monitored as endpoints of antiaging efficacy, were significantly reduced by propolis following UVB exposure in a model of reconstituted skin tissue. In conclusion, propolis protects against the oxidative and photodamaging effects of UVB and could be further explored as a promising agent for developing natural antiaging strategies.

Time dependent response of daunorubicin on cytotoxicity, cell cycle and DNA repair in acute lymphoblastic leukaemia

BackgroundDaunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL). The aim of this study was to explore the kinetics of double strand break (DSB) formation of three ALL cell lines following exposure to daunorubicin and to investigate the effects of daunorubicin on the cell cycle and the protein kinases involved in specific checkpoints following DNA damage and recovery periods.MethodsThree ALL cell lines CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes were examined following 4 h treatment with daunorubicin chemotherapy and 4, 12 and 24 h recovery periods. Cell viability was measured via MTT (3-(4,5-dimethylthiazol-2-yl)-2–5 diphenyltetrazolium bromide) assay, reactive oxygen species (ROS) production by flow cytometry, double stranded DNA breaks by detecting γH2AX levels while stages of the cell cycle were detected following propidium iodide staining and flow cytometry. Western blotting was used to detect specific proteins while RNA was extracted from all cell lines and converted to cDNA to sequence Ataxia–telangiectasia mutated (ATM).ResultsDaunorubicin induced different degrees of toxicity in all cell lines and consistently generated reactive oxygen species. Daunorubicin was more potent at inducing DSB in MOLT-4 and CCRF-CEM cell lines while SUP-B15 cells showed delays in DSB repair and significantly more resistance to daunorubicin compared to the other cell lines as measured by γH2AX assay. Daunorubicin also causes cell cycle arrest in all three cell lines at different checkpoints at different times. These effects were not due to mutations in ATM as sequencing revealed none in any of the three cell lines. However, p53 was phosphorylated at serine 15 only in CCRF-CEM and MOLT-4 but not in SUP-B15 cells. The lack of active p53 may be correlated to the increase of SOD2 in SUP-B15 cells.ConclusionsThe delay in DSB repair and lower sensitivity to daunorubicin seen in the B lymphocyte derived SUP-B15 cells could be due to loss of function of p53 that may be correlated to increased expression of SOD2 and lower ROS production.

Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair

Fanconi anemia (FA) is a cancer predisposition syndrome characterized by congenital abnormalities, bone marrow failure, and hypersensitivity to aldehydes and crosslinking agents. For FA patients, gene editing holds promise for therapeutic applications aimed at functionally restoring mutated genes in hematopoietic stem cells. However, intrinsic FA DNA repair defects may obstruct gene editing feasibility. Here, we report on the CRISPR/Cas9-mediated correction of a disruptive mutation in Fancf. Our experiments revealed that gene editing could effectively restore Fancf function via error-prone end joining resulting in a 27% increased survival in the presence of mitomycin C. In addition, templated gene correction could be achieved after double strand or single strand break formation. Although templated gene editing efficiencies were low (≤6%), FA corrected embryonic stem cells acquired a strong proliferative advantage over non-corrected cells, even without imposing genotoxic stress. Notably, Cas9 nickase activity resulted in mono-allelic gene editing and avoidance of undesired mutagenesis. In conclusion: DNA repair defects associated with FANCF deficiency do not prohibit CRISPR/Cas9 gene correction. Our data provide a solid basis for the application of pre-clinical models to further explore the potential of gene editing against FA, with the eventual aim to obtain therapeutic strategies against bone marrow failure.

Analysis of DNA Damage Biomarkers in Human Leukocytes by PAHs Exposure

Aims: To study the potential genotoxicity of two polynuclear aromatic hydrocarbons (PAHs), exposed cultured human leukocytes in vitro using two types of biomarkers genotoxicity: DNA strand breaks (DNA-SB) and adduct formation (DNA-PAHs).
 Study Design: Human leukocytes were exposed to toxic cultures with different concentrations of anthracene (ANT), phenanthrene (PHE) and benzo(a)pyrene (B(a)P) for 24 hours. Four toxic test groups, PAHs, control group, analytic blank group and standard fluorescence group were considered. 
 Place and Duration of Study: Laboratorio de Citopatología Ambiental, of Departamento de Morfología at Escuela Nacional de Ciencias Biológicas, IPN; between february 2016 and july 2018.
 Methodology: Human leukocytes cells were isolated and cell viability was previously verified by Trypan dye exclusion test. Firstly, the lethal concentrations with neutral red (NR50) assay for each one PAHs was obtained. Then sublethal concentrations range of these toxics for both biomarkers were used. In case of DNA fragmentation, a fluorochrome was used to mark DNA strandbreaks and isolation with alkaline solution finally determined with fluorescence spectroscopy. To test the formation of DNA-PAHs adducts, first of all they were isolated with a solvent system with polarity gradient and finally determined with fluorescence spectroscopy.
 Results: PAHs with 3 aromatic rings showed lethal cytotoxicity, lower in case of the B(a)P with 5 aromatic rings. These results are in contrast with previously reported observations, in which non-adduct with DNA and DNA strandbreaks formation were detected with Anthracene and Phenanthrene while DNA adduction formation and DNA strandbreaks were produced in case of B(a)P.
 Conclusion: Biomarkers may be used as suitable discriminants of genotoxic agents as well as of environmental pollutants with genotoxic potential and for application in studies of environmental risk assessment and in hazardous waste evaluation. The application of both genotoxic biomarkers, DNA strand breaks and production of adducts DNA-PAHs, as genotoxicity assays are quickly and accurate techniques for determining the carcinogenic potential of environmental samples.

Effect of cryopreservation on the formation of DNA double strand breaks in human peripheral blood mononuclear cells

Chemotherapy and radiation therapy are used in malignant oncological diseases to increase the level of DNA double strand breaks (DSBs) in tumor cells. Unrepaired DSBs may either kill a cell or induce terminal arrest. Those DNA damages can be detected by fluorescence imaging of phosphorylated histon e protein H2AX (γH2AX). Peripheral blood mononuclear cells (PBMCs) are easily available human material, which are usually stored by cryopreservation for repeated experiments or postponed analysis. As DNA DSBs can be introduced through several different stressors, it was of interest to investigate the potential impact of cryopreservation, on the formation of DSBs in human PBMCs. The PBMCs were cryopreserved in four different media, containing different proportions of Dulbecco’s Modified Eagle Medium (DMEM), fetal calf serum (FCS) and dimethyl sulfoxide (DMSO) as taken from the literature. Immunofluorescence staining of γH2AX was performed on freshly isolated and cryopreserved PBMCs. A significant reduction on relative γH2AX level, after treatment with 100 μM etoposide (ETP), was observed for cryopreserved cells in medium containing DMEM, which seemed to be the limiting factor on DNA DSB formation. No change was observed for untreated PBMCs. Therefore, DSBs of cryopreserved human PBMCs could be used to investigate the influence of chemotherapeutic drugs or radiation therapy, assuming that the cryopreservation follows a standardized protocol with a tight control of potential DSB inducing stressors.

Genome-wide mapping of 8-oxo-7,8-dihydro-2'-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells.

8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is one of the major DNA modifications and a potent pre-mutagenic lesion prone to mispair with 2′-deoxyadenosine (dA). Several thousand residues of 8-oxodG are constitutively generated in the genome of mammalian cells, but their genomic distribution has not yet been fully characterized. Here, by using OxiDIP-Seq, a highly sensitive methodology that uses immuno-precipitation with efficient anti–8-oxodG antibodies combined with high-throughput sequencing, we report the genome-wide distribution of 8-oxodG in human non-tumorigenic epithelial breast cells (MCF10A), and mouse embryonic fibroblasts (MEFs). OxiDIP-Seq revealed sites of 8-oxodG accumulation overlapping with γH2AX ChIP-Seq signals within the gene body of transcribed long genes, particularly at the DNA replication origins contained therein. We propose that the presence of persistent single-stranded DNA, as a consequence of transcription-replication clashes at these sites, determines local vulnerability to DNA oxidation and/or its slow repair. This oxidatively-generated damage, likely in combination with other kinds of lesion, might contribute to the formation of DNA double strand breaks and activation of DNA damage response.