title: [Induction of robust senescence-associated secretory phenotype in mouse NIH-3T3 cells by mitomycin C].
abstract: Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, β-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 μg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of β-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1β increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of β-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|The Second Clinical School, Guangdong Medical University, Dongguan 523808, China.
authors.forename: Wei-Xing
authors.initials: WX
authors.lastname: Huang
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|The Second Clinical School, Guangdong Medical University, Dongguan 523808, China.
authors.forename: Xiao-Xuan
authors.initials: XX
authors.lastname: Guo
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|The Second Clinical School, Guangdong Medical University, Dongguan 523808, China.
authors.forename: Zhong-Zhi
authors.initials: ZZ
authors.lastname: Peng
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|The Second Clinical School, Guangdong Medical University, Dongguan 523808, China.
authors.forename: Chun-Liang
authors.initials: CL
authors.lastname: Weng
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|The Second Clinical School, Guangdong Medical University, Dongguan 523808, China.
authors.forename: Chun-Yan
authors.initials: CY
authors.lastname: Huang
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical University, Dongguan 523808, China.
authors.forename: Ben-Yan
authors.initials: BY
authors.lastname: Shi
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|The Second Clinical School, Guangdong Medical University, Dongguan 523808, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|School of Laboratory Medicine, Guangdong Medical University, Dongguan 523808, China.
authors.forename: Xiao-Xin
authors.initials: XX
authors.lastname: Liao
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|School of Laboratory Medicine, Guangdong Medical University, Dongguan 523808, China.
authors.forename: Xiao-Yi
authors.initials: XY
authors.lastname: Li
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical University, Dongguan 523808, China.
authors.forename: Hui-Ling
authors.initials: HL
authors.lastname: Zheng
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical University, Dongguan 523808, China.|Institute of Biochemistry & Molecular Biology, Guangdong Medical University, Zhanjiang 524023, China.
authors.forename: Xin-Guang
authors.initials: XG
authors.lastname: Liu
authors.affiliation: Institute of Aging Research, Dongguan Scientific Research Center, Guangdong Medical University, Dongguan 523808, China.|Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Guangdong Medical University, Dongguan 523808, China. xuerongsun@126.com.
authors.forename: Xue-Rong
authors.initials: XR
authors.lastname: Sun
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D016922:Cellular Senescence / Q000187:drug effects*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D005347:Fibroblasts / Q000187:drug effects*; D015850:Interleukin-6 / Q000378:metabolism; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology*; D041681:NIH 3T3 Cells; D010641:Phenotype
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D016207:Cytokines; D015850:Interleukin-6; D016685:Mitomycin