Liao Geng-You, Lee Ming-Ting, Fan Jhen-Jia, Hsiao Pei-Wen, Lee Chun-Sheng, Su Shou-Yi, Hwang Jiuan-Jiuan, Ke Ferng-Chun
Institute of Molecular and Cellular Biology, College of Life Science, National Taiwan University, Taipei 106, Taiwan.
Institute of Physiology, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan.
Biol Open. 2019 May 16;8(5):bio038257. doi: 10.1242/bio.038257.
The purpose of study was to explore the role of glutamine-dependent anaplerosis in cell fate determination (proliferation and senescence) and the potential associated mechanism by employing a pharmacological inhibitor of glutamine-dependent anaplerosis, amino-oxyacetate (AOA). Using the WI38 normal human embryonic fibroblast cell line, we found that exposure to AOA induced mTORC1 inactivation-mTORC2 activation (within day 1), cell cycle arrest (day 2-6) and cellular senescence (day 4-6). These AOA effects were blocked by concomitantly providing anaplerotic factors [α-ketoglutarate (αKG), pyruvate or oxaloacetate], and not affected by ROS scavenger N-acetyl-cysteine (NAC). Moreover, AOA-induced cellular senescence in WI38 cells is associated with elevated protein levels of p53, p21 and p16 and decreased Rb protein level, which was blocked by αKG supplementation. In p16-deficient U2OS human osteosarcoma cells and p16-knockdown WI38 cells, AOA exposure also induced similar effects on cell proliferation, and protein level of P-Rb-S807/811 and Rb. Interestingly, no AOA induction of cellular senescence was observed in U2OS cells, yet was still seen in p16-knockdown WI38 cells accompanied by the presence of p16 antibody-reactive p12. In summary, we disclose that glutamine-dependent anaplerosis is essential to cell growth and closely associated with mTORC1 activation and mTORC2 inactivation, and impedes cellular senescence particularly associated with p16.
本研究的目的是通过使用谷氨酰胺依赖性回补途径的药理学抑制剂氨氧基乙酸（AOA），探讨谷氨酰胺依赖性回补途径在细胞命运决定（增殖和衰老）中的作用以及潜在的相关机制。使用WI38正常人胚胎成纤维细胞系，我们发现暴露于AOA会诱导mTORC1失活 - mTORC2激活（第1天内）、细胞周期停滞（第2 - 6天）和细胞衰老（第4 - 6天）。这些AOA的作用可通过同时提供回补因子[α - 酮戊二酸（αKG）、丙酮酸或草酰乙酸]来阻断，且不受活性氧清除剂N - 乙酰半胱氨酸（NAC）的影响。此外，AOA诱导WI38细胞衰老与p53、p21和p16蛋白水平升高以及Rb蛋白水平降低有关，补充αKG可阻断这种现象。在p16缺陷的U2OS人骨肉瘤细胞和p16敲低的WI38细胞中，暴露于AOA对细胞增殖以及P - Rb - S807/811和Rb蛋白水平也有类似影响。有趣的是，在U2OS细胞中未观察到AOA诱导的细胞衰老，但在p16敲低的WI38细胞中仍可见，且伴有p16抗体反应性p12的存在。总之，我们揭示了谷氨酰胺依赖性回补途径对细胞生长至关重要，且与mTORC1激活和mTORC2失活密切相关，并能阻碍特别是与p16相关的细胞衰老。
