scholarly journals Measuring DNA repair incision activity of mouse tissue extracts towards singlet oxygen-induced DNA damage: a comet-based in vitro repair assay

Mutagenesis ◽  
2011 ◽  
Vol 26 (3) ◽  
pp. 461-471 ◽  
Author(s):  
S. A. S. Langie ◽  
K. M. Cameron ◽  
K. J. Waldron ◽  
K. P. R. Fletcher ◽  
T. von Zglinicki ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Singlet Oxygen ◽  
Mouse Tissue ◽  
Tissue Extracts ◽  
In Vitro Repair

Kaempferol Induces DNA Damage and Inhibits DNA Repair Associated Protein Expressions in Human Promyelocytic Leukemia HL-60 Cells

2015 ◽  
Vol 43 (02) ◽  
pp. 365-382 ◽  
Author(s):  
Lung-Yuan Wu ◽  
Hsu-Feng Lu ◽  
Yu-Cheng Chou ◽  
Yung-Luen Shih ◽  
Da-Tian Bau ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Protein Expression ◽  
Ataxia Telangiectasia ◽  
Repair System ◽  
Dapi Staining ◽  
Viable Cells ◽  
Protein Expressions ◽  
Dose Dependent

Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O6-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60 cells, which may be the factors for kaempferol induced cell death in vitro.

scholarly journals Acquired temozolomide resistance in MGMT-deficient glioblastoma cells is associated with regulation of DNA repair by DHC2

Brain ◽  
2019 ◽  
Vol 142 (8) ◽  
pp. 2352-2366 ◽  
Author(s):  
Guo-zhong Yi ◽  
Guanglong Huang ◽  
Manlan Guo ◽  
Xi’an Zhang ◽  
Hai Wang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Progression Free Survival ◽  
Recurrent Glioblastoma ◽  
Dna Lesions ◽  
Dna Repair Proteins

Abstract The acquisition of temozolomide resistance is a major clinical challenge for glioblastoma treatment. Chemoresistance in glioblastoma is largely attributed to repair of temozolomide-induced DNA lesions by O6-methylguanine-DNA methyltransferase (MGMT). However, some MGMT-deficient glioblastomas are still resistant to temozolomide, and the underlying molecular mechanisms remain unclear. We found that DYNC2H1 (DHC2) was expressed more in MGMT-deficient recurrent glioblastoma specimens and its expression strongly correlated to poor progression-free survival in MGMT promotor methylated glioblastoma patients. Furthermore, silencing DHC2, both in vitro and in vivo, enhanced temozolomide-induced DNA damage and significantly improved the efficiency of temozolomide treatment in MGMT-deficient glioblastoma. Using a combination of subcellular proteomics and in vitro analyses, we showed that DHC2 was involved in nuclear localization of the DNA repair proteins, namely XPC and CBX5, and knockdown of either XPC or CBX5 resulted in increased temozolomide-induced DNA damage. In summary, we identified the nuclear transportation of DNA repair proteins by DHC2 as a critical regulator of acquired temozolomide resistance in MGMT-deficient glioblastoma. Our study offers novel insights for improving therapeutic management of MGMT-deficient glioblastoma.

Drug-induced DNA damage and tumor chemosensitivity.

1990 ◽  
Vol 8 (12) ◽  
pp. 2062-2084 ◽  
Author(s):  
R J Epstein
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Tumor Cell ◽  
Malignant Disease ◽  
Dna Lesions ◽  
Drug Induced ◽  
Cell Subpopulations ◽  
Therapeutic Cell

Cytotoxic drugs act principally by damaging tumor-cell DNA. Quantitative analysis of this interaction provides a basis for understanding the biology of therapeutic cell kill as well as a rational strategy for optimizing and predicting tumor response. Recent advances have made it possible to correlate assayed DNA lesions with cytotoxicity in tumor cell lines, in animal models, and in patients with malignant disease. In addition, many of the complex interrelationships between DNA damage, DNA repair, and alterations of gene expression in response to DNA damage have been defined. Techniques for modulating DNA damage and cytotoxicity using schedule-specific cytotoxic combinations, DNA repair inhibitors, cell-cycle manipulations, and adjunctive noncytotoxic drug therapy are being developed, and critical therapeutic targets have been identified within tumor-cell subpopulations and genomic DNA alike. Most importantly, methods for predicting clinical response to cytotoxic therapy using both in vitro markers of tumor-cell sensitivity and in vivo measurements of drug-induced DNA damage are now becoming a reality. These advances can be expected to provide a strong foundation for the development of innovative cytotoxic drug strategies over the next decade.

The mTOR Inhibitor Everolimus Overcomes CXCR4-Mediated Resistance to HDAC Inhibitor Panobinostat through Inhibition of p21 and Mitosis Regulators, Sensitizing MM Cells to DNA-Damaged Induced Apoptosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 891-891
Author(s):  
Katia Beider ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Evgenia Rosenberg ◽  
Hila Magen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Cell Lines ◽  
Hdac Inhibitor ◽  
Mtor Inhibitor ◽  
Mtor Inhibition ◽  
Induced Apoptosis

Abstract Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Despite the initial efficacious treatment, MM patients often become refractory to common anti-MM drugs, therefore novel therapies are in need. Pan-histone deacetylase (HDAC) inhibitor panobinostat exerts multiple cytotoxic actions in MM cells in vitro, and was approved for the treatment of relapsed/refractory MM in combination with bortezomib and dexamethasone. Although having promising anti-MM properties, panobinostat lacks therapeutic activity as monotherapy. The aim of the current study was to elucidate the mechanisms underlying MM resistance to panobinostat and to define strategies to overcome it. Results: Panobinostat at the low concentrations (IC50 5-30 nM) suppressed the viability in MM cell lines (n=7) and primary CD138+ cells from MM patients (n=8) in vitro. Sensitivity to panobinostat correlated with reduced expression of chemokine receptor CXCR4, while overexpression of CXCR4 or its ligand CXCL12 in RPMI8226 and CAG MM cell lines significantly (p<0.001) increased their resistance to panobinostat, pointing to the role of the CXCR4 axis in HDACi response. Notably, similar expression levels of class I HDACs (HDAC1-3) were detected in MM cells with either low or high CXCR4. Interaction with BM stromal cells that represent the source of CXCL12 also protected MM cells from panobinostat-induced apoptosis, further strengthening a role for CXCR4 downstream pathway. Decreased sensitivity to cytotoxic effect was concomitant with reduced histone (H3K9 and H4K8) acetylation in response to panobinostat treatment. In addition, resistance to HDACi was associated with the reversible G0/G1 cell growth arrest, whereas sensitivity was characterized by apoptotic cell death. Analysis of intra-cellular signaling mediators involved in CXCR4-mediated HDACi resistance revealed the pro-survival AKT/mTOR pathway to be regulated by both CXCR4 over-expression and interaction with BMSCs. Combining panobinostat with mTOR inhibitor everolimus abrogated the resistance and induced synergistic cell death of MM cell lines and primary MM cells, but not of normal mononuclear cells (CI<0.4). This effect was concurrent with the increase in DNA double strand breaks, histone H2AX phosphorylation, loss of Dψm, cytochrome c release, caspase 3 activation and PARP cleavage. The increase in DNA damage upon combinational treatment was not secondary to the apoptotic DNA fragmentation, as it occurred similarly when apoptosis onset was blocked by caspase inhibitor z-VAD-fmk. Kinetics studies also confirmed that panobinostat-induced DNA damage preceded apoptosis induction. Strikingly, combined panobinostat/everolimus treatment resulted in sustained DNA damage and irreversible suppression of MM cell proliferation accompanied by robust apoptosis, in contrast to the modest effects induced by single agent. Gene expression analysis revealed distinct genetic profiles of single versus combined exposures. Whereas panobinostat increased the expression of cell cycle inhibitors GADD45G and p21, co-treatment with everolimus abrogated the increase in p21 and synergistically downregulated DNA repair genes, including RAD21, Ku70, Ku80 and DNA-PKcs. Furthermore, combined treatment markedly decreased both mRNA and protein expression of anti-apoptotic factors survivin and BCL-XL, checkpoint regulator CHK1, and G2/M-specific factors PLK1, CDK1 and cyclin B1, therefore suppressing the DNA damage repair and inhibiting mitotic progression. Given the anti-apoptotic role of p21, the synergistic lethal effect of everolimus could be attributed to its ability to suppress p21 induction by panobinostat ensuing the shift in the DNA damage response toward apoptosis. Conclusions: Collectively, our findings indicate that CXCR4/CXCL12 activity promotes the resistance of MM cells to HDACi with panobinostat through mTOR activation. Inhibition of mTOR by everolimus synergizes with panobinostat by simultaneous suppression of p21, G2/M mitotic factors and DNA repair machinery, rendering MM cells incapable of repairing accumulated DNA damage and promoting their apoptosis. Our results unravel the mechanism responsible for strong synergistic anti-MM activity of dual HDAC and mTOR inhibition and provide the rationale for a novel therapeutic strategy to eradicate MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals MicroRNA regulation of the MRN complex impacts DNA damage, cellular senescence and angiogenic signaling

2017 ◽  
Author(s):  
Cristina Espinosa-Diez ◽  
RaeAnna Wilson ◽  
Namita Chatterjee ◽  
Clayton Hudson ◽  
Rebecca Ruhl ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Targeted Delivery ◽  
Genotoxic Stress ◽  
Telomerase Activity ◽  
Loss Of Function ◽  
Mrn Complex ◽  
Vegf Signaling

AbstractMicroRNAs contribute to biological robustness by buffering cellular processes from external perturbations. Here we report an unexpected link between DNA damage response and angiogenic signaling that is buffered by two distinct microRNAs. We demonstrate that genotoxic stress-induced miR-494 and miR-99b inhibit the DNA repair machinery by targeting the MRE11a-RAD50-NBN (MRN) complex. Functionally, gain and loss of function experiments show that miR-494 and miR-99b affect telomerase activity, activate p21 and Rb pathways and diminish angiogenic sproutingin vitroandin vivo. Genetic and pharmacological disruption of VEGFR-2 signaling and the MRN complex reveal a surprising co-dependency of these pathways in regulating endothelial senescence and proliferation. Vascular-targeted delivery of miR-494 decreases both growth factor-induced and tumor angiogenesis in mouse models. Mechanistically, disruption of the MRN complex induced CD44, a known driver of senescence and regulator of VEGF signaling in addition to suppressing IL-13 a stimulator of VEGF signaling. Our work identifies a putative miR-facilitated mechanism by which endothelial cells can be insulated against VEGF signaling to facilitate the onset of senescence and highlight the potential of targeting DNA repair to disrupt pathological angiogenesis.

scholarly journals A CRISPR-associated factor Csa3a regulates DNA damage repair in Crenarchaeon Sulfolobus islandicus

2020 ◽  
Vol 48 (17) ◽  
pp. 9681-9693
Author(s):  
Zhenzhen Liu ◽  
Mengmeng Sun ◽  
Jilin Liu ◽  
Tao Liu ◽  
Qing Ye ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Genomic Dna ◽  
Cell Aggregation ◽  
Gene Promoters ◽  
Dna Damages ◽  
Associated Factor ◽  
Sulfolobus Islandicus ◽  
Repair Systems

Abstract CRISPR−Cas system provides acquired immunity against invasive genetic elements in prokaryotes. In both bacteria and archaea, transcriptional factors play important roles in regulation of CRISPR adaptation and interference. In the model Crenarchaeon Sulfolobus islandicus, a CRISPR-associated factor Csa3a triggers CRISPR adaptation and activates CRISPR RNA transcription for the immunity. However, regulation of DNA repair systems for repairing the genomic DNA damages caused by the CRISPR self-immunity is less understood. Here, according to the transcriptome and reporter gene data, we found that deletion of the csa3a gene down-regulated the DNA damage response (DDR) genes, including the ups and ced genes. Furthermore, in vitro analyses demonstrated that Csa3a specifically bound the DDR gene promoters. Microscopic analysis showed that deletion of csa3a significantly inhibited DNA damage-induced cell aggregation. Moreover, the flow cytometry study and survival rate analysis revealed that the csa3a deletion strain was more sensitive to the DNA-damaging reagent. Importantly, CRISPR self-targeting and DNA transfer experiments revealed that Csa3a was involved in regulating Ups- and Ced-mediated repair of CRISPR-damaged host genomic DNA. These results explain the interplay between Csa3a functions in activating CRISPR adaptation and DNA repair systems, and expands our understanding of the lost link between CRISPR self-immunity and genome stability.

Ordered Intracellular Reca-Dna Assemblies

2000 ◽  
Vol 6 (S2) ◽  
pp. 860-861
Author(s):  
S. G. Wolf ◽  
S. Levin-Zaidman ◽  
D. Frenkiel-Krispin ◽  
E. Shimoni ◽  
I. Sabanay ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Electron Micrographs ◽  
Reca Protein ◽  
Sos Response ◽  
Structural Features ◽  
Wild Type ◽  
E Coli ◽  
Dna Damaging Agents

The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. We find that induction of the SOS system in wild-type E. coli bacteria results in fast and massive intracellular coaggregation of RecA and DNA into lateral assemblies, which comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in-vitro RecA-DNA are consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.Bacterial chromatin is demarcated in electron micrographs of metabolically active cells as amorphous ribosome-free spaces that are irregularly spread over the cytoplasm (Fig. A). Wild-type E. coli cells exposed to DNA-damaging agents that induce the SOS response reveal a strikingly different morphology (Fig. B).

scholarly journals Symmetric activity of DNA polymerases at and recruitment of exonuclease ExoR and of PolA to the Bacillus subtilis replication forks

2019 ◽  
Vol 47 (16) ◽  
pp. 8521-8536 ◽  
Author(s):  
Rogelio Hernández-Tamayo ◽  
Luis M Oviedo-Bocanegra ◽  
Georg Fritz ◽  
Peter L Graumann
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Bacillus Subtilis ◽  
Single Molecule ◽  
Dna Polymerases ◽  
Replication Forks ◽  
Single Molecule Level ◽  
Time Dynamics ◽  
Bacterium Bacillus

AbstractDNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5′ overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.

P–437 The ovaries of transgender men indicate effects of high dose testosterone on the primordial and early growing follicle pool

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
E Bailie ◽  
M Maidarti ◽  
R Hawthorn ◽  
S Jack ◽  
N Watson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Histological Analysis ◽  
High Dose ◽  
Follicle Growth ◽  
Repair Capacity ◽  
Testosterone Undecanoate ◽  
The Impact ◽  
Transgender Men

Abstract Study question Does high-dose testosterone therapy affect the stage distribution, morphological health and DNA damage repair capacity of human ovarian follicles and their survival in vitro? Summary answer Testosterone exposure is associated with reduced follicle growth activation, reduced follicle health and increased DNA damage: these further deteriorate after six days of culture. What is known already: Androgens have diverse actions within the ovary, however, there is a lack of information regarding the long-term effects of high-dose testosterone on ovarian function and reproductive potential. Cumulus-oocyte complexes recovered from transgender men have been successfully matured in-vitro but little is known regarding the impact of this gender affirming endocrine therapy on the primordial follicle pool. Study design, size, duration: Whole ovaries were obtained from four transgender men aged 25–36 years with informed consent at oophorectomy. All patients had received 1000mg testosterone undecanoate intramuscularly at 12–16 week intervals for a minimum of 4 years pre-operatively. Cortical tissues were dissected into small pieces (≈1x1x0.5mm) and either immediately fixed for histological analysis or cultured for 6 days. Testosterone-treated ovaries were compared to cortical biopsies from age-matched healthy women obtained at caesarean section (n = 4, age 26–36). Participants/materials, setting, methods: Follicle number, classification of developmental stage and morphology were evaluated by histological analysis of ovarian cortical tissue from day 0 and 6 days post culture. Immunohistochemical analysis included γH2AX as a marker of DNA damage, and meiotic recombination 11 (MRE11), ataxia telangiectasia mutated (ATM) and Rad51 as DNA repair proteins. A total of 3802 follicles from testosterone exposed and 878 from control ovaries were analysed. Main results and the role of chance: At day 0 (D0), transgender tissue had a higher proportion of non-growing follicles (92.7±1.7%) compared to control (85.4±6.2%, p &lt; 0.05) but a lower proportion of morphologically healthy follicles (non-growing 59%, primary 61%, secondary 36%; vs 83%, 75%, 80% in controls, all p &lt; 0.005). After 6 days in culture, the proportion of growing follicles increased (51.3% vs 46.5%) but follicle health further declined (all stages p &lt; 0.005). DNA damage was assessed by expression of γH2AX. At D0, the proportion of oocytes showing DNA damage was significantly higher in transgender non-growing follicles (48.1±12.5%, vs 12.3±0.25%, p &lt; 0.005). After culture, γH2AX expression increased in both transgender (p &lt; 0.005) and controls (p &lt; 0.005) but remained higher in transgender oocytes (non-growing 72.2%, primary 71.7% vs 27.3%, 46.2%, all p &lt; 0.05). At D0, there was no difference in expression of DNA repair enzymes ATM and RAD51 between transgender and control oocytes, and increased expression of MRE11 in control non-growing follicles (p &lt; 0.05). Post-culture, there was a significant increase in ATM expression in transgender non-growing oocytes compared to control (98.5% vs 77.8%, p &lt; 0.05) and a less marked decline in RAD51 expression(p &lt; 0.05). The expression of MRE–11 in control non-growing oocytes dramatically declined (100% to 58.2%, p &lt; 0.05), unlike in transgender tissue where expression was comparable to D0. Limitations, reasons for caution A large number of follicles have been analysed, but only from a small number of ovaries. DNA damage at D0 and after 6 days of culture may not reflect DNA damage and repair capacity at later stages of follicle growth. The effect of duration of testosterone treatment was not investigated. Wider implications of the findings: These data indicate that high circulating concentrations of testosterone have previously unrecognised effects on the primordial and small-growing follicles of the ovary. These results may have implications for transgender men receiving gender-affirming therapy prior to considering pregnancy or fertility preservation measures. Trial registration number n/a

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