caa a22 4500 606233768 CHVBK 20210128101233.0 cr unu---uuuuu 210128e20150601xx s 000 0 eng 10.1007/s11010-015-2388-0 doi (NATIONALLICENCE)springer-10.1007/s11010-015-2388-0 Rac1 GTPase-deficient HeLa cells present reduced DNA repair, proliferation, and survival under UV or gamma irradiation [Elektronische Daten] [Gisele Espinha, Juliana Osaki, Yuli Magalhaes, Fabio Forti] Rac1 GTPase controls essential cellular functions related to the cytoskeleton, such as motility and adhesion. Rac1 is overexpressed in many tumor cells, including breast cancers, where it is also involved in the proliferation and checkpoint control necessary for the cell's recovery after exposure to ionizing radiation. However, its role in DNA damage and repair remains obscure in other tumor cells and under different genotoxic conditions. Here, we compare HeLa cells with mutants exogenously expressing a dominant-negative Rac1 (HeLa-Rac1-N17) by their responses to DNA damage induced by gamma or UV radiation. In HeLa cells, these treatments led to increased levels of active Rac1 (Rac1-GTP) and of stress fibers, with a diminished ability to migrate compared to untreated cells. However, the reduction of Rac1-GTP in Rac1-N17-deficient clones resulted in much higher levels of polymerized stress fibers accompanied by a strong impairment of cell migration, even after both radiation treatments. With regard to proliferation and genomic stability, dominant-negative Rac1 cells were more sensitive to gamma and UV radiation, exhibiting reduced proliferation and survival consistent with increased DNA damage and delayed or reduced DNA repair observed in this Rac1-deficient clone. The DNA damage response, as indicated by pH2AX and pChk1 levels, was increased in HeLa cells but was not effectively triggered in the Rac1-N17 clone after radiation treatment, which is likely the main cause of DNA damage accumulation. These data suggest that Rac1 GTPase plays an important role in signaling and contributes to the sensitivity of cervical cancer cells under UV or gamma radiation treatments. Springer Science+Business Media New York, 2015 Small Rho GTPases nationallicence Rac1 GTPase nationallicence DNA damage response and repair nationallicence HeLa cells nationallicence UV and gamma radiation nationallicence 6-4PP : Pyrimidine (6-4) pyrimidone photoproduct nationallicence 8-oxoG : 8-Oxo-7,8-dihydroguanine nationallicence AML : Acute myeloid leukemia nationallicence ATM : Ataxia telangiectasia mutated kinase nationallicence ATR : Ataxia telangiectasia mutated-related kinase nationallicence BSA : Bovine serum albumin nationallicence CPD : Cyclobutane pyrimidine dimmer nationallicence DAPI : Dilactate 4′,6-di-amino-2-phenylindole nationallicence DDR : DNA damage response nationallicence DME : Dulbecco's modified Eagle's medium nationallicence DMSO : Dimethyl sulfoxide nationallicence DNA : Deoxyribonucleic acid nationallicence DSB : Double-strand break nationallicence DTT : Dithiothreitol nationallicence EDTA : Ethylenediaminetetraacetic acid nationallicence FBS : Fetal bovine serum nationallicence FDR : False-discovery rate algorithm nationallicence G2/M : Gap 2/mitosis transition of cell cycle nationallicence GAP : GTPase-activating proteins nationallicence GDP : Guanosine diphosphate nationallicence GEF : Guanine exchange factor nationallicence GO : Gene ontology nationallicence GTP : Guanosine triphosphate nationallicence Gy : Gray nationallicence HR : Homologous recombination nationallicence IPTG : Isopropyl β-d thiogalactopyranoside nationallicence J/m2 : Joule/meters2 nationallicence MAPK : Mitogen-activated protein kinase nationallicence MEF : Mouse embryonic fibroblast nationallicence MLC : Myosin light chain nationallicence NER : Nucleotide excision repair nationallicence NHEJ : Non-homologous end-joining nationallicence Nox : NADPH-oxidase complex nationallicence PAGE : Polyacrylamide gel electrophoresis nationallicence PAK : p21-activating kinase nationallicence PBD-GST : PAK1-binding domain linked to glutathione S-transferase nationallicence PBS : Phosphate buffered saline nationallicence PIKK : Phosphatidylinositol 3-kinase-related kinase protein nationallicence PMSF : Phenylmethylsulfonyl fluoride nationallicence PPPI : Physical protein-protein interaction nationallicence RNS : Reactive nitrogen species nationallicence ROS : Reactive oxygen species nationallicence SCGE : Single-cell gel electrophoresis nationallicence SDS : Sodium dodecyl sulfate nationallicence SSB : Single-strand break nationallicence UV : Ultraviolet radiation nationallicence Espinha Gisele Laboratory of Signaling in Biomolecular Systems, Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes, 748 - Bl.09i, Sl.922, Cidade Universitária, CEP: 05508-900, São Paulo, SP, Brazil aut Osaki Juliana Laboratory of Signaling in Biomolecular Systems, Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes, 748 - Bl.09i, Sl.922, Cidade Universitária, CEP: 05508-900, São Paulo, SP, Brazil aut Magalhaes Yuli Laboratory of Signaling in Biomolecular Systems, Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes, 748 - Bl.09i, Sl.922, Cidade Universitária, CEP: 05508-900, São Paulo, SP, Brazil aut Forti Fabio Laboratory of Signaling in Biomolecular Systems, Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes, 748 - Bl.09i, Sl.922, Cidade Universitária, CEP: 05508-900, São Paulo, SP, Brazil aut Molecular and Cellular Biochemistry Springer US; http://www.springer-ny.com 404/1-2(2015-06-01), 281-297 0300-8177 404:1-2<281 2015 404 11010 https://doi.org/10.1007/s11010-015-2388-0 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s11010-015-2388-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Espinha Gisele Laboratory of Signaling in Biomolecular Systems, Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes, 748 - Bl.09i, Sl.922, Cidade Universitária, CEP: 05508-900, São Paulo, SP, Brazil aut NATIONALLICENCE 700 1- Osaki Juliana Laboratory of Signaling in Biomolecular Systems, Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes, 748 - Bl.09i, Sl.922, Cidade Universitária, CEP: 05508-900, São Paulo, SP, Brazil aut NATIONALLICENCE 700 1- Magalhaes Yuli Laboratory of Signaling in Biomolecular Systems, Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes, 748 - Bl.09i, Sl.922, Cidade Universitária, CEP: 05508-900, São Paulo, SP, Brazil aut NATIONALLICENCE 700 1- Forti Fabio Laboratory of Signaling in Biomolecular Systems, Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Av. Prof. Lineu Prestes, 748 - Bl.09i, Sl.922, Cidade Universitária, CEP: 05508-900, São Paulo, SP, Brazil aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Springer US; http://www.springer-ny.com 404/1-2(2015-06-01), 281-297 0300-8177 404:1-2<281 2015 404 11010