scholarly journals Effects of quin2 acetoxymethyl ester on H2O2-induced DNA single-strand breakage in mammalian cells: H2O2-concentration-dependent inhibition of damage and additive protective effect with the hydroxyl-radical scavenger dimethyl sulphoxide

1995 ◽  
Vol 305 (1) ◽  
pp. 181-185 ◽  
Author(s):  
B E Sandström
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Single Strand ◽  
Dimethyl Sulphoxide ◽  
Radical Scavenger ◽  
Dna Breaks ◽  
Acetoxymethyl Ester ◽  
Dna Breakage ◽  
Strand Breakage

The cell-membrane-permeable calcium probe quin2 acetoxymethyl ester (quin2 AM) was ineffective, in comparison with o-phenanthroline, in protecting cells against H2O2-induced DNA single-strand breakage at H2O2 concentrations of about, and higher than, 0.5 mM. The present study shows that quin2 actually potentiated intracellular DNA damage at high H2O2 concentrations. H2O2-induced DNA breakage appeared within 5 min after exposure, and quin2 affected the induction of DNA breaks at both 0 degree C and 37 degrees C. Aurintricarboxylic acid, an endonuclease inhibitor, or a decrease in extracellular Ca2+, did not reduce DNA damage. These facts strongly suggest that the breaks were not produced by a Ca(2+)-dependent nuclease. We showed previously that, in the presence of Fe3+ and H2O2, quin2 strongly potentiated the formation of oxidizing species as well as plasmid DNA breakage, and, as could be expected for a transition-metal chelator, quin2 inhibited the Fenton reaction when Cu2+ was tested instead of Fe3+ [Sandström, Granström and Marklund (1994) Free Radicals Biol. Med. 16, 177-185]. In the present work with cultured cells, titration with quin2 AM showed that, despite the fact that Cu2+ has a three-to-four-orders-of-magnitude higher affinity for quin2 than has Fe3+, both inhibition and potentiation of H2O2-induced DNA damage occurred at quin2 AM concentrations of about 100 nM. Thus inhibition appeared not to involve Cu2+. The combination of quin2 AM and dimethyl sulphoxide (DMSO) gave an additive effect on H2O2-induced DNA damage compared with the effect of quin2 AM or DMSO alone, whereas the combination of o-phenanthroline and DMSO gave about the same effect as o-phenanthroline alone. In conclusion, our results do not support a role for Ca2+ in the inhibiting effect of quin2 on H2O2-induced DNA damage. Instead, it is likely that inhibition and potentiation by quin2 involves interaction with Fe ions.

scholarly journals Protection of sperm DNA against oxidative stress in vivo by accessory sex gland secretions in male hamsters

Reproduction ◽  
2002 ◽  
pp. 491-499 ◽  
Author(s):  
H Chen ◽  
MP Cheung ◽  
PH Chow ◽  
AL Cheung ◽  
W Liu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Comet Assay ◽  
Prostate Gland ◽  
Single Strand ◽  
Ventral Prostate ◽  
Control Group ◽  
Dna Breakage ◽  
Accessory Glands ◽  
Sperm Dna ◽  
Epididymal Spermatozoa

Reactive oxygen species scavengers present in male accessory sex gland secretions might afford antioxidant protection to sperm DNA. This study was conducted to determine whether accessory sex gland secretions protect the genome and function of spermatozoa against oxidative damage in the uterus. Male golden hamsters were divided into four experimental groups: (i) all accessory sex glands removed; (ii) ampullary glands removed; (iii) ventral prostate gland removed and (iv) sham-operated controls. Ejaculated spermatozoa recovered from uteri 15-30 min after mating with experimental males and caput and cauda epididymal spermatozoa obtained from intact males were incubated in 0-20 mmol NADPH l(-1) for 2 h. These spermatozoa and untreated uterine spermatozoa were processed for two types of comet assay (single cell gel electrophoresis): alkaline comet assay (pH > 13) which revealed single-strand DNA breakage and neutral comet assay (pH 9) which revealed double-strand DNA breakage. In comparison with the sham-operated controls, spermatozoa that had not been exposed to accessory sex gland secretions had a higher incidence and more extensive single-strand DNA damage with increasing concentrations of NADPH. Spermatozoa from hamsters without ampullary glands and from hamsters without the ventral prostate glands were similar to those of the control group. After incubation with NADPH, the capacity of spermatozoa from hamsters without accessory glands and from sham-operated controls to fuse with oocytes in vitro was reduced. However, only hamsters without accessory glands showed a negative correlation between single-strand DNA damage and sperm-oocyte fusion. Cauda epididymal spermatozoa were less susceptible to NADPH treatment compared with caput epididymal spermatozoa. The results of the present study showed that male accessory sex gland secretions can preserve the integrity of the sperm genome.

scholarly journals Induction of gadd153 mRNA by nutrient deprivation is overcome by glutamine

1999 ◽  
Vol 341 (1) ◽  
pp. 225-231 ◽  
Author(s):  
Qihong HUANG ◽  
Serrine S. LAU ◽  
Terrence J. MONKS
Keyword(s):  
Dna Damage ◽  
Mrna Expression ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Nutrient Deprivation ◽  
Cellular Energy ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular ◽  
Renal Proximal Tubular ◽  
Gadd153 Expression

The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of gadd153 mRNA was investigated in renal proximal tubular epithelial cells (LLC-PK1) cultured in a nutrient- and serum-deprived medium. The addition of glutamine alone to LLC-PK1 cells cultured in Earl's balanced salt solution (EBSS) is sufficient to suppress gadd153 mRNA expression, and the removal of only glutamine from Dulbecco's modified Eagle's medium (DMEM) is also sufficient to induce gadd153 mRNA expression. Consistent with these findings, the inhibition of glutamine utilization with acivicin and 6-diazo-5-oxo-L-norleucine (DON) in cells grown in a glutamine-containing medium effectively induces gadd153 expression. Glutamine can be used as an energy source in cultured mammalian cells. However, it is unlikely that deficits in cellular energy stores (ATP) are coupled to gadd153 mRNA expression, because concentrations of ATP, UTP and GTP are all elevated in EBSS-exposed cells, and the addition of α-oxoglutarate to cells grown in EBSS has no effect on gadd153 mRNA expression. In contrast, concentrations of CTP decline substantially in EBSS and glutamine-deprived DMEM-cultured cells. Glutamine also serves as a precursor for the synthesis of protein and DNA. The addition of glutamine to cells grown in EBSS partly restores CTP concentrations. The addition of pyrimidine ribonucleosides (cytidine and uridine) to LLC-PK1 cells also restores CTP concentrations, in a manner commensurate with their relative abilities to overcome gadd153 expression. Finally, glutamine does not completely suppress DNA damage-induced gadd153 expression, suggesting that multiple signalling pathways lead to the expression of gadd153 mRNA under conditions of nutrient deprivation and DNA damage.

scholarly journals Engineered proteins detect spontaneous DNA breakage in human and bacterial cells

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Chandan Shee ◽  
Ben D Cox ◽  
Franklin Gu ◽  
Elizabeth M Luengas ◽  
Mohan C Joshi ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Cytosine Deaminase ◽  
Bacterial Cells ◽  
Bacteriophage Mu ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Breakage ◽  
Genomic Changes

Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP—labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells.

scholarly journals Synergy between SIRT1 and SIRT6 helps recognize DNA breaks and potentiate the DNA damage response and repair

2020 ◽  
Author(s):  
Fanbiao Meng ◽  
Minxian Qian ◽  
Bin Peng ◽  
Xiaohui Wang ◽  
Linyuan Peng ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Cultured Cells ◽  
Synergistic Action ◽  
Dna Breaks ◽  
Spatiotemporal Regulation ◽  
Double Strand Dna Breaks ◽  
Damage Response ◽  
Defective Dna Repair

SummaryThe DNA damage response (DDR) is a highly orchestrated process but how double-strand DNA breaks (DSBs) are initially recognized is unclear. Here, we show that polymerized SIRT6 deacetylase recognizes DSBs and potentiates the DDR. First, SIRT1 deacetylates SIRT6 at residue K33, which is important for SIRT6 polymerization and mobilization toward DSBs. Then, K33-deacetylated SIRT6 anchors to γH2AX, allowing its retention on and subsequent remodeling of local chromatin. We show that a K33R mutation that mimics hypoacetylated SIRT6 can rescue defective DNA repair as a result of SIRT1 deficiency in cultured cells. These data highlight the synergistic action between SIRTs in the spatiotemporal regulation of the DDR and DNA repair.

scholarly journals Nonhomologous-End-Joining Factors Regulate DNA Repair Fidelity during Sleeping Beauty Element Transposition in Mammalian Cells

2003 ◽  
Vol 23 (23) ◽  
pp. 8505-8518 ◽  
Author(s):  
Stephen R. Yant ◽  
Mark A. Kay
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Mammalian Cells ◽  
Donor Site ◽  
Protective Role ◽  
Sleeping Beauty ◽  
Nonhomologous End Joining ◽  
Dna Breaks ◽  
End Joining ◽  
Transposon Insertion

ABSTRACT Herein, we report that the DNA-dependent protein kinase (DNA-PK) regulates the DNA damage introduced during Sleeping Beauty (SB) element excision and reinsertion in mammalian cells. Using both plasmid- and chromosome-based mobility assays, we analyzed the repair of transposase-induced double-stranded DNA breaks in cells deficient in either the DNA-binding subunit of DNA-PK (Ku) or its catalytic subunit (DNA-PKcs). We found that the free 3′ overhangs left after SB element excision were efficiently and accurately processed by the major Ku-dependent nonhomologous-end-joining pathway. Rejoining of broken DNA molecules in the absence of Ku resulted in extensive end degradation at the donor site and greatly increased the frequency of recombination with ectopic templates. Therefore, the major DNA-PK-dependent DNA damage response predominates over more-error-prone repair pathways and thereby facilitates high-fidelity DNA repair during transposon mobilization in mammalian cells. Although transposable elements were not found to be efficiently circularized after transposase-mediated excision, DNA-PK deficiency supported more-frequent transposase-mediated element insertion than was found in wild-type controls. We conclude that, based on its ability to regulate excision site junctional diversity and transposon insertion frequency, DNA-PK serves an important protective role during transpositional recombination in mammals.

Fast Neutron and X-Ray Induced Single Strand DNA Breaks in Cultured Mammalian Cells

Radiology ◽  
1976 ◽  
Vol 119 (2) ◽  
pp. 459-461 ◽  
Author(s):  
Alfred J. Moss ◽  
Max L. Baker ◽  
Richard M. Prior ◽  
Erik A. Erichsen ◽  
William A. Nagle ◽  
...  
Keyword(s):  
Fast Neutron ◽  
Mammalian Cells ◽  
Single Strand ◽  
Dna Breaks ◽  
X Ray

scholarly journals Iron requirement for cellular DNA damage and growth inhibition by hydrogen peroxide and bleomycin

1994 ◽  
Vol 302 (3) ◽  
pp. 655-664 ◽  
Author(s):  
K Radtke ◽  
F A Lornitzo ◽  
R W Byrnes ◽  
W E Antholine ◽  
D H Petering
Keyword(s):  
Dna Damage ◽  
Growth Inhibition ◽  
Euglena Gracilis ◽  
Culture Media ◽  
Control Cell ◽  
Cell Types ◽  
Single Strand ◽  
Intracellular Iron ◽  
Iron Deficient ◽  
Strand Breakage

Studies with Euglena gracilis and HL-60 cells have assessed the need for intracellular iron in the mechanisms of inhibition of cell growth and DNA damage by H2O2 and bleomycin. Cell culture media were directly depleted of iron in order to deprive cells of nutrient iron. Major pools of cellular iron were reduced in both cell types. Nevertheless, iron bound in e.s.r.-observable haem protein and ribonucleotide diphosphate reductase in HL-60 cells was not decreased. In both control cell populations, there was a concentration-dependent reduction in proliferation and cell survival caused by H2O2. In comparison, the proliferation rates of both iron-deficient cell types were significantly less sensitive to H2O2. H2O2 caused concentration-dependent single-strand breakage in DNA in control HL-60 and Euglena gracilis cells. Iron deficiency reduced the amount of strand breaks in HL-60 cells at each concentration of H2O2 used. Single-strand breakage caused by H2O2 in Euglena gracilis was a direct function of the concentration of iron in which the cells had been grown. Growth inhibition and both single- and double-strand DNA damage caused by bleomycin were substantially reduced or eliminated in iron-deficient cells. Copper bleomycin behaved like metal-free bleomycin when assayed for the capacity to cause DNA damage in iron-normal and iron-deficient HL-60 cells. In contrast, iron bleomycin was equally active under the two conditions in these cells.

scholarly journals Expression of the HPV18/E6 oncoprotein induces DNA damage

2017 ◽  
Author(s):  
Elva I. Cortés Gutiérrez ◽  
Catalina Garcí­a-Vielma ◽  
Adriana Aguilar-Lemarroy ◽  
Veronica Vallejo-Ruí­z ◽  
Patricia Piña-Sánchez ◽  
...  
Keyword(s):  
Dna Damage ◽  
Hela Cells ◽  
Dna Hybridization ◽  
Dna Breaks ◽  
Dna Breakage ◽  
Hpv E6 ◽  
E6 Oncoprotein ◽  
Normal Expression ◽  
Hpv18 E6

This study investigated possible variations in DNA damage in HeLa cells with silenced expression of the HPV/E6 oncogene compared with HeLa cells with normal expression of the E6 oncogene using the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) technique and a whole human genome DNA probe. The variable levels of DNA breaks present were measured quantitatively using image analysis after whole-genome DNA hybridization. HeLa cells with silenced expression of the HPV18/E6 oncogene showed a significant decrease in DNA damage compared with parental cells with normal expression of the E6 oncogene. These results were confirmed by alkaline comet assay. In conclusion, we demonstrated a decrease in DNA damage in HeLa clones associated with low expression of the HPV/E6 oncogene. The significance of this decrease regarding the HPV life cycle and carcinogenesis requires further exploration.

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