• Molecules and Cells
• /
• 제42권7호
• /
• pp.546-556
• /
• 2019

Protein phosphatase 4 (PP4) is a crucial protein complex that plays an important role in DNA damage response (DDR), including DNA repair, cell cycle arrest and apoptosis. Despite the significance of PP4, the mechanism by which PP4 is regulated remains to be elucidated. Here, we identified a novel PP4 inhibitor, protein phosphatase 4 inhibitory protein (PP4IP) and elucidated its cellular functions. PP4IP-knockout cells were generated using the CRISPR/Cas9 system, and the phosphorylation status of PP4 substrates (H2AX, KAP1, and RPA2) was analyzed. Then we investigated that how PP4IP affects the cellular functions of PP4 by immunoprecipitation, immunofluorescence, and DNA double-strand break (DSB) repair assays. PP4IP interacts with PP4 complex, which is affected by DNA damage and cell cycle progression and decreases the dephosphorylational activity of PP4. Both overexpression and depletion of PP4IP impairs DSB repairs and sensitizes cells to genotoxic stress, suggesting timely inhibition of PP4 to be indispensable for cells in responding to DNA damage. Our results identify a novel inhibitor of PP4 that inhibits PP4-mediated cellular functions and establish the physiological importance of this regulation. In addition, PP4IP might be developed as potential therapeutic reagents for targeting tumors particularly with high level of PP4C expression.

• Molecular & Cellular Toxicology
• /
• 제1권2호
• /
• pp.99-105
• /
• 2005

Perchloroethylene (tetrachloroethylene, PCE), a dry cleaning and degreasing solvent, can enter ground-water through accidental leak or spills. PCE can be degraded to trichloroethylene (TCE), 1, 1-dichloroethylene (DCE) and vinyl chloride (VC) as potential bio-product. These compounds have been reported that they can cause clinical diseases and cytotoxicity. However, only a little genotoxic information of these compounds has been known. In this study, we investigated DNA single strand breaks of PCE, TCE, DCE and VC by single cell gel electrophoresis assay, (comet assay) which is a sensitive, reliable and rapid method for DNA single strand breaks with mouse lymphoma L5178Y cells. From these results, $37.5\;{\mu}g/ml$ of PCE, $189\;{\mu}g/ml$ of TCE and $56.4\;{\mu}g/ml$ of DCE were revealed significant DNA damages in the absence of S-9 metabolic activation system meaning direct-acting mutagen. And in the presence of S-9 metabolic activation system, $41.5\;{\mu}g/ml$ of PCE, $328.7\;{\mu}g/ml$ of TCE and $949\;{\mu}g/ml$ of DCE were induced significant DNA damage. In the case of VC, it was revealed a significant DNA damage in the presence of S-9 metabolic activation system. Therefore, we suggest that chloroethylene compounds (PCE, TCE, DCE and VC) may be induced the DNA damage in a mammalian cell.

• Journal of Applied Biological Chemistry
• /
• 제43권3호
• /
• pp.156-160
• /
• 2000

The possible mechanism of the DNA strand breaking activity of the hot-water extract of Fructus Chaenomelis (dried fruit of Chaenomeles sinensis) in a closed circular duplex replica form DNA (RFI DNA) was studied through agarose gel electrophresis under various conditions. Induction of DNA strand scission by the hot-water extract of C. sinensis occurred in dose and time-dependent manners. $Cu^{2+}$ was indispensable for the induction of DNA strand breakage. Exogeneous chelating agents inhibited the DNA breaking activity, conforming the catalytic action of $Cu^{2+}$ on generation of free radicals responsible for oxidative damage. Antioxidant enzymes and some radical scavengers were used to investigate the major radical species triggering the DNA strand scission, demonstrating that a highest inhibitory activity was found in the presence of catalase, while less in the presence of tiron (a scavenger for superoxide radical), 2-aminoethyl-isothiuroniumbromide-HBr, cysteamine (scavengers for hydroxyl radical), and 1,4-diazabicyclo [2,2,2] octane (a scavenger for singlet oxygen) in decreasing order. The findings implied that oxygen radical species generated in presence of transition divalent cation during the oxidation of some compounds contained in the hot-water extract of C. sinensis is mainly responsible for inducing genotoxicity.

• 대한본초학회지
• /
• 제22권1호
• /
• pp.7-12
• /
• 2007

Objectives : In Alzheimer's disease(AD), free radical oxidative stress caused by amyloid beta-peptide may lead to DNA damage, neuronal dysfunction, neurotoxicity and cell death, Hwangryunhaedok-tang(HHT) is traditionally used for the treatment of pyrogenetic diseases. To develop a new anti-AD drug from natural herb, HHT was selected and extracted in this study. Methods : The antioxidant activities of HHT water extract powder were examined by hydroxyl radical-induced DNA strand nicking assay, and antioxidative enzymes expression assay in H4IIE cell. In addition, HHT was examined for the inhibitory effect on the acetylcholinesterase(AChE) using by Ellman's coupled assay. Results: The HHT exhibit DNA protective effect in the hydroxyl radical-induced DNA Strand nicking assay, mRNA expression of superoxide dismutase and glutathione peroxidase were recovered at a normal level by HHT treatment in H4IIE cell. Furthermore, water extract of HHT showed inhibitory effect on AChE activity. Conclusion : These results suggest that HHT may be effective in delaying and preventing AD progression related to the free radical-induced DNA damage and AChE activity.

• 한국양식학회지
• /
• 제16권3호
• /
• pp.196-201
• /
• 2003

Sessile organisms such as the oyster Crassostrea gigas have been given much attention as a potential biomonitoring indicator to assess the impact of toxicants on aquatic organism. In this study, we exposed cells isolated from gill of oyster (Crassostrea gigas) to hydrogen peroxide in vitro. In addition oysters were in vivo exposed to pyrene and benzo(a)pyrene at various concentrations for 2 weeks. Comet assay was used to detect DNA single strand breaks and to investigate the application of this technique as a tool for aquatic biomonitoring. Hydrogen peroxide increased DNA single strand break with increasing concentration after 30 minutes exposure in vitro. Pyrene and benzo(a)pyrene caused DNA damage only at very high concentration (100 $\mu\textrm{g}$/L or 1000 $\mu\textrm{g}$/L) at two week exposure in vivo. DNA damage was relatively higher at hemocyte than at gill. It suggested that metabolized PAHs are transferred to hemolymph from digestive gland which have a relatively high enzyme activity, and attacked the DNA of hemocyte, while gill accumulated PAHs without degrading them to their metabolites due to low enzyme activity at gill. Both in vitro and in vivo exposure experiments showed that the comet assay is an effective tool on screening whether the organism are exposed to genotoxic contaminants.

• Molecules and Cells
• /
• 제40권2호
• /
• pp.143-150
• /
• 2017

Homologous recombination (HR) is necessary for maintenance of genomic integrity and prevention of various mutations in tumor suppressor genes and proto-oncogenes. Rad51 and Rad54 are key HR factors that cope with replication stress and DNA breaks in eukaryotes. Rad51 binds to single-stranded DNA (ssDNA) to form the presynaptic filament that promotes a homology search and DNA strand exchange, and Rad54 stimulates the strand-pairing function of Rad51. Here, we studied the molecular dynamics of Rad51 and Rad54 during the cell cycle of HeLa cells. These cells constitutively express Rad51 and Rad54 throughout the entire cell cycle, and the formation of foci immediately increased in response to various types of DNA damage and replication stress, except for caffeine, which suppressed the Rad51-dependent HR pathway. Depletion of Rad51 caused severe defects in response to postreplicative stress. Accordingly, HeLa cells were arrested at the G2-M transition although a small amount of Rad51 was steadily maintained in HeLa cells. Our results suggest that cell cycle progression and proliferation of HeLa cells can be tightly controlled by the abundance of HR proteins, which are essential for the rapid response to postreplicative stress and DNA damage stress.

• The Korean Journal of Physiology and Pharmacology
• /
• 제13권5호
• /
• pp.343-348
• /
• 2009

53BP1 is an important genome stability regulator, which protects cells against double-strand breaks. Following DNA damage, 53BP1 is rapidly recruited to sites of DNA breakage, along with other DNA damage response proteins, including ${\gamma}$-H2AX, MDC1, and BRCA1. The recruitment of 53BP1 requires a tandem Tudor fold which associates with methylated histones H3 and H4. It has already been determined that the majority of DNA damage response proteins are phosphorylated by ATM and/or ATR after DNA damage, and then recruited to the break sites. 53BP1 is also phosphorylated at several sites, like other proteins after DNA damage, but this phosphorylation is not critically relevant to recruitment or repair processes. In this study, we evaluated the functions of phosphor-53BP1 and the role of the BRCT domain of 53BP1 in DNA repair. From our data, we were able to detect differences in the phosphorylation patterns in Ser25 and Ser1778 of 53BP1 after neocarzinostatin-induced DNA damage. Furthermore, the foci formation patterns in both phosphorylation sites of 53BP1 also evidenced sizeable differences following DNA damage. From our results, we concluded that each phosphoryaltion site of 53BP1 performs different roles, and Ser1778 is more important than Ser25 in the process of DNA repair.

• Animal cells and systems
• /
• 제9권2호
• /
• pp.79-85
• /
• 2005

Oxidized abasic residues arise as a major class of DNA damage by a variety of agents involving free radical attack and oxidation of deoxyribose sugar components. 2-deoxyribonolactone (dL) is a C1'-oxidized abasic lesion implicated in DNA strand scission, mutagenesis, and covalent DNA-protein cross-link (DPC). We show here that mammalian cell-free extract give rise to stable DPC formation that is specifically mediated by dL residue. When a duplex DNA containing dL at the site-specific position was incubated with cell-free extracts of Po ${\beta}-proficient$ and -deficient mouse embryonic fibroblast cells, the formation of major dL-mediated DPC was dependent on the presence of DNA polymerase (Pol) ${\beta}$. Formation of dL-specific DPC was also observed with histones and FEN1 nuclease, although the reactivity in forming dL-mediated DPC was significantly higher with Pol ${\beta}$ than with histones or FEN1. DNA repair assay with a defined DPC revealed that the dL lesion once cross-linked with Pol ${\beta}$ was resistant to nucleotide excision repair activity of cell-free extract. Analysis of nucleotide excision repair utilizing a model DNA substrate containing a (6-4) photoproduct suggested that excision process for DPC was inhibited because of DNA single-strand incision at 5' of the lesion. Consequently DPC mediated by dL lesion may not be readily repaired by DNA excision repair pathway but instead function as unusual DNA damage causing a prolonged DNA strand break and trapping of the major base excision repair enzyme.

• Environmental Analysis Health and Toxicology
• /
• 제20권4호통권51호
• /
• pp.333-341
• /
• 2005

In order to know whether polychaetes could be used as an appropriate organism for the detection of genotoxicity, DNA strand breaks were evaluated in blood cells of a nereidae worm (Perinereis aibuhitensis) exposed to various aquatic chemical pollutants (e.g. Cd, Pb, Pyrene, Benaor[a]pyrene). Hydrogen peroxide increased DNA strand breaks up to the highest concentration (10 $\mu$M). Higher concentration than 0.1 $\mu$M showed a significantly more DNA damage than control. Cadmium and lead also showed higher DNA damage than control, over 1.0 and 1 $\mu$g/L, respectively. In case of pyrene, DNA damage was detected even at 0.001 $\mu$g/L. However, DNA damage decreased due to apoptosis at the highest concentration of pyrene and Pb. This study suggested that the polythaetous blood cells could be used effectively for screening genotoxic contaminants in the environment.

• 한국식품위생안전성학회지
• /
• 제31권1호
• /
• pp.1-7
• /
• 2016

본 연구의 목적은 NIH3T3와 HepG2 세포에서 에탄올 유도 세포독성 및 유전독성에 대하여 녹차엑기스(GTE)와 epigallocatechin-3-gallate (EGCG)의 보호작용을 평가하는데 있다. 세포생존율은 MTT assay를 실시하였으며 DNA 손상도는 Comet assay로 실시한 결과 에탄올은 농도의존적인 세포독성과 유전독성을 나타내었다. 한편 GTE와 EGCG는 에탄올 유도 세포독성 및 DNA 손상에 대하여 유의성 있는 억제효과를 나타내었으며 DPPH시험과 LDL oxidation 및 8OH-2'dG 생성시험에서 항산화효과를 나타내었다. 한편 녹차성분 함유 시판 리큐르주도 순수 에탄올에 비하여 세포독성억제 및 DNA 손상억제효과를 나타내었다. 이상의 시험결과 GTE와 함유 EGCG는 항산화성 유전독성억제기전을 통한 에탄올독성저감 물질로 판단된다.