The cellular responses of autophagy, apoptosis, and 5-methylcytosine level in zebrafish cells upon nutrient deprivation stress

Here we reported the stress responses of nutrient deprivation and extended observation of autophagy, apoptosis, and DNA methylation in zebrafish embryonic fibroblast (ZF4) cells. Our results showed that serum deprivation resulted in the changes of cell shape and adherent ability, the suppressed cell growth and viability, and the inhibited proliferation and cell cycle. Besides, the appearance of lysosome and autophagosome/autolysosome with significantly increased expression of mRNAs (ulk1a, becn1, atg12, sqstm1, maplc3, and lamp1) and proteins (Atg12, Becn1, Sqstm1, and Lamp1) indicate the autophagic activity was boosted at initial stage but relatively weakened at 48 h of serum starvation. When autophagy no longer mitigate for the stress, cell apoptosis detected by the mRNA expression of caspases, Bcl-2/Bax expression, and Annexin V/PI was gradually enhanced to execute the death plan upon prolonged starvation process. Furthermore, the methyl group metabolism was increased in accordance with autophagic activity and was suppressed by enhanced apoptotic activity. These data suggested that the recycle activity induced by autophagy could compensate the substrates and reactions of DNA transmethylation, which obviously increased 5-methylcytosine (5 mC) level in ZF4 cells. In summary, our results discovered the cellular responses under prolonged serum starvation stress and elaborated the switch from autophagy to apoptosis and corresponding correlation with 5 mC level changes in teleost fish in vitro. (C) 2019 Elsevier Ltd. All rights reserved.

在此，我们报道了营养剥夺的应激反应，并对斑马鱼胚胎成纤维细胞（ZF4）中的自噬、凋亡和DNA甲基化进行了长期观察。我们的结果显示，血清剥夺导致细胞形状和贴壁能力改变，细胞生长和活力受到抑制，增殖和细胞周期也被抑制。此外，溶酶体和自噬体/自溶酶体的出现，以及mRNA（ulk1a、becn1、atg12、sqstm1、maplc3和lamp1）和蛋白质（Atg12、Becn1、Sqstm1和Lamp1）表达显著增加，表明自噬活性在血清饥饿初期增强，但在48小时时相对减弱。当自噬不再缓解应激时，通过半胱天冬酶的mRNA表达、Bcl - 2/Bax表达以及膜联蛋白V/碘化丙啶检测到的细胞凋亡在长期饥饿过程中逐渐增强，以执行死亡程序。此外，甲基代谢随着自噬活性增加而增加，并因凋亡活性增强而受到抑制。这些数据表明，自噬诱导的循环活动可以补偿DNA转甲基化的底物和反应，这明显提高了ZF4细胞中5 - 甲基胞嘧啶（5mC）的水平。总之，我们的结果揭示了在长期血清饥饿应激下的细胞反应，并阐述了硬骨鱼体外从自噬到凋亡的转换以及与5mC水平变化的相应相关性。（C）2019爱思唯尔有限公司。保留所有权利。