Posen Yehudit, Kalchenko Vyacheslav, Seger Rony, Brandis Alexander, Scherz Avigdor, Salomon Yoram
Department of Biological Regulation, Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel.
J Cell Sci. 2005 May 1;118(Pt 9):1957-69. doi: 10.1242/jcs.02323. Epub 2005 Apr 19.
Reactive oxygen species (ROS) comprise a group of noxious byproducts of oxidative processes which participate in the induction of many common diseases. However, understanding their role in the regulation of normal physiological redox signaling is currently evolving. Detailed study of the dynamic functions of ROS within the biological milieu is difficult because of their high chemical reactivity, short lifetime, minute concentrations and cytotoxicity at high concentrations. In this study, we show that increasing intracellular ROS levels, set off by controlled in situ photogeneration of a nontoxic bacteriochlorophyll-based sensitizer initiate responses in cultured melanoma cells. Using hydroethidine as detector, we determined light-dependent generation of superoxide and hydroxyl radicals in cell-free and cell culture models. Monitoring the ROS-induced responses revealed individual and differential behavior of protein kinases [p38, mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and Akt] as well as effects on the subcellular distribution of phosphorylated p38. Furthermore, alterations in morphology and motility and effects on cell viability as a function of time and photosensitizer doses were observed. Following mild ROS challenge, enzymatic and cellular changes were observed in the majority of the cells, without inducing extensive cell death. However, upon vigorous ROS challenge, a similar profile of the overall responses was observed, terminating in cell death. This study shows that precisely controlled photogeneration of ROS can provide simple, fine-tuned, noninvasive manipulation of ROS-sensitive cellular responses ranging from individual enzymes to gross behavior of target cells. The observations made with this tool enable a dynamic and causal correlation, presenting a new alternative for studying the role of ROS in cellular redox signaling.
活性氧（ROS）是氧化过程产生的一组有害副产物，参与多种常见疾病的诱发。然而，目前人们对其在正常生理氧化还原信号调节中的作用的理解正在不断发展。由于ROS具有高化学反应性、短寿命、低浓度以及高浓度时的细胞毒性，因此很难在生物环境中详细研究其动态功能。在本研究中，我们发现通过基于无毒细菌叶绿素的敏化剂的原位光生作用来提高细胞内ROS水平，可引发培养的黑色素瘤细胞的反应。使用氢乙啶作为检测器，我们在无细胞和细胞培养模型中测定了超氧化物和羟基自由基的光依赖性生成。监测ROS诱导的反应揭示了蛋白激酶[p38、丝裂原活化蛋白激酶（MAPK）、细胞外信号调节激酶（ERK）、c-Jun氨基末端激酶（JNK）和Akt]的个体和差异行为，以及对磷酸化p38亚细胞分布的影响。此外，还观察到细胞形态和运动性的改变以及细胞活力随时间和光敏剂剂量的变化。在轻度ROS刺激后，大多数细胞中观察到酶和细胞的变化，但未诱导广泛的细胞死亡。然而，在强烈的ROS刺激下，观察到了类似的总体反应特征，最终导致细胞死亡。这项研究表明，精确控制的ROS光生作用可以提供对ROS敏感细胞反应的简单、微调、非侵入性操作，范围从单个酶到靶细胞的总体行为。使用该工具进行的观察能够建立动态的因果关系，为研究ROS在细胞氧化还原信号传导中的作用提供了一种新的选择。
