Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii pr. 4, St. Petersburg, 194064, Russia.
Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii pr. 4, St. Petersburg, 194064, Russia; Peter the Great St. Petersburg Polytechnic University, Polytechnicheskaya st. 29, St. Petersburg, 195251, Russia.
Redox Biol. 2022 Apr;50:102245. doi: 10.1016/j.redox.2022.102245. Epub 2022 Jan 26.
Application of genetically encoded biosensors of redox-active compounds promotes the elaboration of new methods for investigation of intracellular redox activities. Previously, we have developed a method to measure quantitatively the intracellular concentration of hydrogen peroxide (HO) in living cells using genetically encoded biosensor HyPer. In the present study, we refined the method and applied it for comparing the antioxidant system potency in human cells of different phenotypes by measuring the gradient between the extracellular and cytoplasmic HO concentrations under conditions of HO-induced external oxidative stress. The measurements were performed using cancer cell lines (K-562 and HeLa), as well as normal human cells - all expressing HyPer in the cell cytoplasm. As normal cells, we used three isogenic lines of different phenotypes - mesenchymal stem/stromal cells (MSCs), induced pluripotent stem cells (iPSCs) derived from MSCs by reprogramming, and differentiated iPSC progenies with the phenotype resembling precursory MSCs. When exposing cells to exogenous HO, we showed that at low oxidative loads (<50 μM of HO) the gradient depended on extracellular HO concentration. At high loads (>50 μM of HO), which caused the exhaustion of thioredoxin activity in the cell cytoplasm, the gradient stabilized, pointing out that it is the functional status of the thioredoxin-depended enzymatic system that drives the dependence of the HO gradient on the oxidative load in human cells. At high HO concentrations, the cytoplasmic HO level in cancer cells was found to be several hundred times lower than the extracellular one. At the same time, in normal cells, extracellular-to-intracellular gradient amounted to thousands of times. Upon reprogramming, the potency of cellular antioxidant defense increased. In contrast, differentiation of iPSCs did not result in the changes in antioxidant system activity in the cell cytoplasm, assuming that intensification of the HO-detoxification processes is inherent to a period of early human development.
应用氧化还原活性化合物的基因编码生物传感器促进了细胞内氧化还原活性新方法的研究。先前，我们开发了一种使用基因编码生物传感器 HyPer 定量测量活细胞内过氧化氢 (HO) 浓度的方法。在本研究中，我们改进了该方法，并应用该方法在 HO 诱导的外部氧化应激条件下，通过测量细胞外和细胞质 HO 浓度之间的梯度，比较不同表型的人细胞中的抗氧化系统效力。该测量使用了癌细胞系 (K-562 和 HeLa)，以及在细胞质中表达 HyPer 的正常人类细胞。作为正常细胞，我们使用了三种不同表型的同基因系 - 间充质干细胞 (MSCs)、由 MSCs 重编程得到的诱导多能干细胞 (iPSCs)，以及具有类似于前体 MSCs 表型的分化 iPSC 后代。当将细胞暴露于外源性 HO 时，我们表明在低氧化负荷 (<50 μM 的 HO) 下，梯度取决于细胞外 HO 浓度。在高负荷 (>50 μM 的 HO) 下，细胞细胞质中的硫氧还蛋白活性耗尽，梯度稳定，表明是硫氧还蛋白依赖性酶系统的功能状态驱动了人细胞中 HO 梯度对氧化负荷的依赖性。在高 HO 浓度下，癌细胞质中的 HO 水平比细胞外低几百倍。同时，在正常细胞中，细胞外到细胞内的梯度达到几千倍。重编程后，细胞抗氧化防御的效力增加。相反，iPSC 的分化不会导致细胞质中抗氧化系统活性的变化，这表明 HO 解毒过程的强化是人类早期发育的固有特性。
