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GNG11 (G-Protein Γ Subunit 11) Suppresses Cell Growth with Induction of Reactive

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GNG11 (G-Protein Γ Subunit 11) Suppresses Cell Growth with Induction of Reactive Biochemistry and Cell Biology GNG11 (G-protein γ subunit 11) suppresses cell growth with induction of reactive oxygen species and abnormal nuclear morphology in human SUSM1 cells Journal: Biochemistry and Cell Biology Manuscript ID bcb-2016-0248.R1 Manuscript Type: Article Date Submitted by the Author: 02-Mar-2017 Complete List of Authors: Takauji, Yuki; Yokohama City University Kudo, Ikuru; Yokohama City University En, Atsuki; DraftYokohama City University Matsuo, Ryo; Yokohama City University Hossain, Mohammad; Primeasia University Nakabayashi, Kazuhiko; National Center for Child Health and Development Miki, Kensuke; Yokohama City University Fujii, Michihiko; Yokohama City University Ayusawa, Dai; Yokohama City University Please Select from this Special N/A Issues list if applicable: GNG11, cellular senescence, cellular immortalization, nuclear shape, Keyword: reactive oxygen species https://mc06.manuscriptcentral.com/bcb-pubs Page 1 of 33 Biochemistry and Cell Biology 1 GNG11 (G-protein γ subunit 11) suppresses cell growth with induction of reactive 2 oxygen species and abnormal nuclear morphology in human SUSM-1 cells 3 4 Yuki Takauji1,5, Ikuru Kudo1,5, Atsuki En1, Ryo Matsuo1, Mohammad Nazir Hossain3, Kazuhiko 5 Nakabayashi4, Kensuke Miki1,2, Michihiko Fujii1* and Dai Ayusawa1,2 6 7 1Graduate school of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, 8 Yokohama, Kanagawa 236-0027, JapanDraft 9 2Ichiban Life Corporation, 1-1-7 Horai-cho, Naka-ku, Yokohama, Kanagawa 231-0048, Japan 10 3Department of Biochemistry, Primeasia University, 9 Banani C/A Banani, Dhaka 1213, Bangladesh 11 4Department of Maternal-Fetal Biology, National Center for Child Health and Development, Tokyo 12 157-8535, Japan 13 5Both authors contributed equally to this work. 14 15 *To whom correspondence should be addressed. 16 Michihiko Fujii, Tel&Fax: +81-45-787-2213; E-mail address: [email protected] 17 1 https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 2 of 33 1 Abstract 2 Enforced expression of GNG11, G-protein γ subunit 11, induces cellular senescence in normal 3 human diploid fibroblasts. We here examined the effect of the expression of GNG11 on the growth of 4 immortalized human cell lines, and found that it suppressed the growth of SUSM-1 cells, but not of 5 HeLa cells. We then compared these two cell lines to understand the molecular basis for the action of 6 GNG11. We found that expression of GNG11 induced the generation of reactive oxygen species 7 (ROS) and abnormal nuclear morphology in SUSM-1 cells but not in HeLa cells. Increased ROS 8 generation by GNG11 would likely be causedDraft by the down-regulation of the antioxidant enzymes in 9 SUSM-1 cells. We also found that SUSM-1 cells, even under normal culture conditions, showed 10 higher levels of ROS and higher incidence of abnormal nuclear morphology than HeLa cells, and 11 that abnormal nuclear morphology was relevant to the increased ROS generation in SUSM-1 cells. 12 Thus, SUSM-1 and HeLa cells showed differences in the regulation of ROS and nuclear morphology, 13 which might account for their different responses to the expression of GNG11. Then, SUSM-1 cells 14 may provide a unique system to study the regulatory relationship between ROS generation, nuclear 15 morphology, and G-protein signaling. 16 Key words: GNG11; cellular senescence; cellular immortalization; nuclear shape; reactive oxygen 17 species; 18 2 https://mc06.manuscriptcentral.com/bcb-pubs Page 3 of 33 Biochemistry and Cell Biology 1 Introduction 2 Normal types of human somatic cell undergo terminal growth arrest after a limited number of 3 cell divisions in vitro, a phenomenon termed cellular senescence or replicative senescence (Hayflick 4 and Moorhead 1961). Cellular senescence can also be induced by various means such as reactive 5 oxygen species (ROS), DNA damages, cell cycle perturbations, chromatin destabilization, etc 6 (Michishita et al. 1999; Young and Smith 2000). Cells can escape from cellular senescence by 7 mutational events and acquire an ability to grow indefinitely. For instance, mutations in tumor 8 suppressor genes such as p53, RB, and INK4a/ARFDraft are well-known to help cells escape from cellular 9 senescence. Then, immortalized cell lines have different genetic backgrounds, and the difference in 10 their genetic backgrounds would be causative for their distinct responses to various stimuli or 11 treatments. 12 We have shown that 5-bromodeoxyuridine (BrdU), an analogue of thymidine, effectively 13 induces cellular senescence in various types of cell (Michishita et al. 1999). BrdU is a thymidine 14 analogue and thus exerts its effects upon incorporation into genomic DNA in the place of thymidine. 15 Since BrdU decondenses heterochromatin through destabilization or disruption of nucleosome 16 positioning (Miki et al. 2008; Miki et al. 2010; Zakharov et al. 1974), dysregulation of 17 heterochromatin seems to be involved in the induction of cellular senescence. Heterochromatin is 18 tethered to the nuclear envelope and has roles in the organization of the nuclear envelope. To analyze 3 https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 4 of 33 1 the molecular basis for the action of BrdU in cellular senescence, we screened for the genes 2 up-regulated in BrdU-induced senescent cells, and identified GNG11, γ-subunit 11 of the 3 heterotrimeric G-protein (Hossain et al. 2006; Suzuki et al. 2001). The heterotrimeric G-proteins are 4 composed of the Gα, Gβ, and Gγ subunits, and the Gα and Gβγ subunits work synergistically or 5 independently to transduce signals upon receptor activation (Clapham and Neer 1997). Gβγ subunits 6 reside not only in the plasma membrane but also in endosomes, endoplasmic reticulum, the Golgi 7 apparatus, mitochondria, and nuclei (Khan et al. 2013). GNG11 (Gγ11) forms a complex with the Gβ1 8 subunit and shuttles between the plasmaDraft membrane and the Golgi apparatus (Cho et al. 2011; Saini 9 et al. 2010); however, the function of GNG11 has been largely unidentified. 10 We have previously shown that enforced expression of GNG11 suppresses the growth of 11 normal human diploid fibroblast TIG-7 cells (Hossain et al. 2006). We then examined whether 12 expression of GNG11 suppresses the growth of immortalized cell lines. For this, we employed 13 human immortal SUSM-1 and HeLa cells, and found that expression of GNG11 suppressed the 14 growth of SUSM-1 cells, but not of HeLa cells. We then compared SUSM-1 cells with HeLa cells to 15 understand the mechanisms for the growth-suppressive effect of GNG11 in SUSM-1 cells, and found 16 that ROS and nuclear morphology were regulated differently in SUSM-1 cells than in HeLa cells, 17 and these differences may account for the different responses to the expression of GNG11 in 18 SUSM-1 cells and HeLa cells. 4 https://mc06.manuscriptcentral.com/bcb-pubs Page 5 of 33 Biochemistry and Cell Biology 1 Materials and Methods 2 Cell culture 3 Cervical tumor-derived HeLa cells were obtained from the Japanese Collection of Research 4 Bioresources (JCRB). SUSM-1 cells were established from fetal human diploid fibroblast (AD387) 5 by repeated mutagen treatments and were kindly provided by Dr. Namba, M (Namba et al. 1993). 6 Cells were cultured in plastic dishes (Thermo Scientific, Nunc) containing Dulbecco's modified 7 Eagle's Medium (DMEM) (Nissui Seiyaku) supplemented with 5% fetal calf serum (Cell culture 8 bioscience) at 37°C in 5% CO2 and 95%Draft humidity as described previously (Michishita et al. 1999). 9 Paraquat (Sigma-Aldrich) and BrdU (Wako) were added to the cells at the concentrations of 5-40 10 µM and 50 µM, respectively. 11 12 Colony formation assay 13 To determine the survival of cells, appropriate numbers of cells (1 x 103 - 1 x 104) were plated 14 on 30- or 60-mm dishes, and allowed to grow for 1-2 weeks. To observe colony formation, cells 15 were fixed with methanol and stained with Coomassie Brilliant Blue (CBB, Bio-Rad), and subjected 16 to photography. 17 18 Plasmids 5 https://mc06.manuscriptcentral.com/bcb-pubs Biochemistry and Cell Biology Page 6 of 33 1 The plasmid encoding YFP-tagged GNG11 was kindly provided by Dr. Gautam (Washington 2 University school of Medicine) (Cho et al. 2011; Saini et al. 2010). Construction of the plasmid that 3 expresses GNG11 or GFP from the human cytomegalovirus promoter was previously described 4 (Hossain et al. 2006). 5 6 DNA transfection 7 Plasmids (10 µg) were introduced into cells (106 cells) by electroporation with a 8 high-efficiency electroporator (type NEPA21,Draft Nepa Gene), and appropriate numbers of the cells 9 (1×103-106 cells) were seeded on dishes or cover slips for further analyses. 10 11 Senescence-associated ß-Galactosidase assay 12 Cells were fixed in 2% formaldehyde/ 0.2% glutaraldehyde at room temperature for 5 min, and 13 incubated at 37˚C with a fresh staining solution [1 mg/ml of 5-bromo-4-chloro-3-indolyl 14 ß-D-galactoside, 40 mM citric acid-sodium phosphate (pH 6.0), 5 mM potassium ferricyanide, 5 mM 15 potassium ferrocyanide, 150 mM NaCl, and 2 mM MgC12] as previously described (Michishita et al. 16 1999). 17 18 Assay of ROS 6 https://mc06.manuscriptcentral.com/bcb-pubs Page 7 of 33 Biochemistry and Cell Biology 1 The cells were washed twice with PBS (phosphate buffered saline), and incubated in serum 2 free medium supplemented with 5 µM of a ROS-sensitive fluorescence probe, 3 2’7’-dichloro-fluorescein diacetate (H2DCFDA, Molecular probes) for 30 min at 37˚C in dark. Then, 4 cells were washed twice with PBS, and the fluorescence signals were photographed with a 5 fluorescence microscope (IX-70, Olympus) equipped with a standard filter set for fluorescein.
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