Deficiency of Grx1 leads to high sensitivity of HeLaS3 cells to oxidative stress via excessive accumulation of intracellular oxidants including ROS.
Identifieur interne : 000081 ( Main/Exploration ); précédent : 000080; suivant : 000082Deficiency of Grx1 leads to high sensitivity of HeLaS3 cells to oxidative stress via excessive accumulation of intracellular oxidants including ROS.
Auteurs : Tingyi Zhao [Japon] ; Qiu-Mei Zhang-Akiyama [Japon]Source :
- Free radical research [ 1029-2470 ] ; 2020.
Abstract
Oxidative stress is often initiated by excess reactive oxygen species (ROS) production, resulting in macromolecular damage, which is implicated in many disease states. Glutaredoxin 1 (Grx1) is an antioxidant enzyme that plays an important role in redox signaling and redox homeostasis. In the present study, we generated HeLaS3 cell lines deficient in Grx1 by the CRISPR/CAS9 system to clarify how Grx1 affects the physiological activities of HeLaS3 cells to respond to oxidative stress. First, the survival assay revealed that Grx1-deficient HeLaS3 cells were more sensitive to γ-ray irradiation, heat shock and H2O2 exposure than HeLaS3 wild-type cells. Next, the intracellular redox state was investigated using a fluorescent probe (2'-7'dichlorofluorescin diacetate), and the oxidized state of total proteins and a peroxidase Prx2 were measured by Western blot analysis. Exposure to γ-ray irradiation, heat shock and H2O2 significantly induced more accumulation of intracellular oxidants including ROS and higher levels of oxidized proteins in Grx1-deficient HeLaS3 cells. Furthermore, MitoSox Red staining demonstrated that Grx1 deficiency causes a higher level of oxidants production in mitochondria. Moreover, Grx1-deficient HeLaS3 cells had a higher cytochrome c level and higher apoptosis rate (Annexin-V/FITC and EthD-III staining assay) upon oxidative stress. These results suggested that Grx1 deficiency lead to mitochondrial redox homeostasis disruption and apoptotic cell death upon oxidative stress. In addition, the results of proliferation assay and MitoTracker staining assay (multinuclear cell formation rate) suggested that oxidative stress exposure inhibits cell proliferation maybe by affecting cytoplasmic division in Grx1-deficient HeLaS3 cells.
DOI: 10.1080/10715762.2020.1819994
PubMed: 32892658
Affiliations:
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Glutaredoxin 1 (Grx1) is an antioxidant enzyme that plays an important role in redox signaling and redox homeostasis. In the present study, we generated HeLaS3 cell lines deficient in Grx1 by the CRISPR/CAS9 system to clarify how Grx1 affects the physiological activities of HeLaS3 cells to respond to oxidative stress. First, the survival assay revealed that Grx1-deficient HeLaS3 cells were more sensitive to γ-ray irradiation, heat shock and H<sub>2</sub>O<sub>2</sub> exposure than HeLaS3 wild-type cells. Next, the intracellular redox state was investigated using a fluorescent probe (2'-7'dichlorofluorescin diacetate), and the oxidized state of total proteins and a peroxidase Prx2 were measured by Western blot analysis. Exposure to γ-ray irradiation, heat shock and H<sub>2</sub>O<sub>2</sub> significantly induced more accumulation of intracellular oxidants including ROS and higher levels of oxidized proteins in Grx1-deficient HeLaS3 cells. Furthermore, MitoSox Red staining demonstrated that Grx1 deficiency causes a higher level of oxidants production in mitochondria. Moreover, Grx1-deficient HeLaS3 cells had a higher cytochrome c level and higher apoptosis rate (Annexin-V/FITC and EthD-III staining assay) upon oxidative stress. These results suggested that Grx1 deficiency lead to mitochondrial redox homeostasis disruption and apoptotic cell death upon oxidative stress. In addition, the results of proliferation assay and MitoTracker staining assay (multinuclear cell formation rate) suggested that oxidative stress exposure inhibits cell proliferation maybe by affecting cytoplasmic division in Grx1-deficient HeLaS3 cells.</div></front></TEI><pubmed><MedlineCitation Status="Publisher" Owner="NLM"><PMID Version="1">32892658</PMID><DateRevised><Year>2020</Year><Month>10</Month><Day>06</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1029-2470</ISSN><JournalIssue CitedMedium="Internet"><PubDate><Year>2020</Year><Month>Sep</Month><Day>21</Day></PubDate></JournalIssue><Title>Free radical research</Title><ISOAbbreviation>Free Radic Res</ISOAbbreviation></Journal><ArticleTitle>Deficiency of Grx1 leads to high sensitivity of HeLaS3 cells to oxidative stress via excessive accumulation of intracellular oxidants including ROS.</ArticleTitle><Pagination><MedlinePgn>1-21</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1080/10715762.2020.1819994</ELocationID><Abstract><AbstractText>Oxidative stress is often initiated by excess reactive oxygen species (ROS) production, resulting in macromolecular damage, which is implicated in many disease states. Glutaredoxin 1 (Grx1) is an antioxidant enzyme that plays an important role in redox signaling and redox homeostasis. In the present study, we generated HeLaS3 cell lines deficient in Grx1 by the CRISPR/CAS9 system to clarify how Grx1 affects the physiological activities of HeLaS3 cells to respond to oxidative stress. First, the survival assay revealed that Grx1-deficient HeLaS3 cells were more sensitive to γ-ray irradiation, heat shock and H<sub>2</sub>O<sub>2</sub> exposure than HeLaS3 wild-type cells. Next, the intracellular redox state was investigated using a fluorescent probe (2'-7'dichlorofluorescin diacetate), and the oxidized state of total proteins and a peroxidase Prx2 were measured by Western blot analysis. Exposure to γ-ray irradiation, heat shock and H<sub>2</sub>O<sub>2</sub> significantly induced more accumulation of intracellular oxidants including ROS and higher levels of oxidized proteins in Grx1-deficient HeLaS3 cells. Furthermore, MitoSox Red staining demonstrated that Grx1 deficiency causes a higher level of oxidants production in mitochondria. Moreover, Grx1-deficient HeLaS3 cells had a higher cytochrome c level and higher apoptosis rate (Annexin-V/FITC and EthD-III staining assay) upon oxidative stress. These results suggested that Grx1 deficiency lead to mitochondrial redox homeostasis disruption and apoptotic cell death upon oxidative stress. In addition, the results of proliferation assay and MitoTracker staining assay (multinuclear cell formation rate) suggested that oxidative stress exposure inhibits cell proliferation maybe by affecting cytoplasmic division in Grx1-deficient HeLaS3 cells.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Zhao</LastName><ForeName>Tingyi</ForeName><Initials>T</Initials><AffiliationInfo><Affiliation>Laboratory of Stress Response Biology, Graduate School of Science, Kyoto University, Kyoto, Japan.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Zhang-Akiyama</LastName><ForeName>Qiu-Mei</ForeName><Initials>QM</Initials><AffiliationInfo><Affiliation>Laboratory of Stress Response Biology, Graduate School of Science, Kyoto University, Kyoto, Japan.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2020</Year><Month>09</Month><Day>21</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>Free Radic Res</MedlineTA><NlmUniqueID>9423872</NlmUniqueID><ISSNLinking>1029-2470</ISSNLinking></MedlineJournalInfo><CitationSubset>IM</CitationSubset><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">Glutaredoxin 1</Keyword><Keyword MajorTopicYN="N">oxidative stress</Keyword><Keyword MajorTopicYN="N">reactive oxygen species</Keyword><Keyword MajorTopicYN="N">redox homeostasis</Keyword><Keyword MajorTopicYN="N">γ-ray irradiation</Keyword></KeywordList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="pubmed"><Year>2020</Year><Month>9</Month><Day>8</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2020</Year><Month>9</Month><Day>8</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2020</Year><Month>9</Month><Day>7</Day><Hour>5</Hour><Minute>25</Minute></PubMedPubDate></History><PublicationStatus>aheadofprint</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">32892658</ArticleId><ArticleId IdType="doi">10.1080/10715762.2020.1819994</ArticleId></ArticleIdList></PubmedData></pubmed><affiliations><list><country><li>Japon</li></country><region><li>Région du Kansai</li></region><settlement><li>Kyoto</li></settlement><orgName><li>Université de Kyoto</li></orgName></list><tree><country name="Japon"><region name="Région du Kansai"><name sortKey="Zhao, Tingyi" sort="Zhao, Tingyi" uniqKey="Zhao T" first="Tingyi" last="Zhao">Tingyi Zhao</name></region><name sortKey="Zhang Akiyama, Qiu Mei" sort="Zhang Akiyama, Qiu Mei" uniqKey="Zhang Akiyama Q" first="Qiu-Mei" last="Zhang-Akiyama">Qiu-Mei Zhang-Akiyama</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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