ROS Induces Mitotic Arrest By Activating Spindle Assembly Checkpoint

Reactive oxygen species(ROS) is a kind of oxygen-containing substance with strong oxidation function including hydrogen peroxide(H2O2), superoxide anion and hydroxyl radical. Usually, people will associate ROS with diseases and aging process because of its capability of oxidizing many components in cells including DNA, lipid and protein, what’s more, the oxidation of these substances will eventually lead to cell death and canceration. Many studies reported that ROS is related to many diseases including ischemic reperfusion injury, atherosclerosis, cancer and chronic inflammation caused by hormone deficiency. ROS is the normal metabolic by-product in cell and it must not be only associated with diseases. In recent years, increasing evidence showed that ROS can play a role as a signal molecule in the some signal pathways in cell. In this paper we preliminarily discuss the function of ROS in mitosis and our study has proposed the function of ROS(main H2O2) in activating SAC for the first time.The cell proliferation process is a cycle and this cycle is divided into the period of mitosis and interphase. During interphase, mainly matter accumulation is conducted as the preparation of cell mitosis. Mitosis is the most important life activity during the eukaryotic cell cycle, to be specific, cells undergoes mitosis to realize cell proliferation. When the chromosome is divided into two daughter cells, the stability of genetic material can be maintained. Cell proliferation is one of the most important features in cell life activities and in order to make it smooth, there exist a series of adjustment and control mechanism within the cells to make sure that the whole process is completed under strict surveillance without any error. Spindle Assembly Checkpoint(SAC) is present in the transitional stage between the premetaphase to metaphase of mitosis while its function is to ensure that all the chromosomes can be correctly captured by spindle fibers before the mitotic metaphase. During the process of mitosis prophase to its late stage, through the monitoring of SAC, when all the chromosomes have been correctly captured and all the work has been properly arranged, cells can successfully enter the mitosis anaphase and finally smoothly finish the whole mitosis phase. After the generation of SAC signal, it exerts effects on molecules Cdc20, SAC protein Bu Rd1, Bu Rd3 and Mad2 combined with Cdc20 to form the mitotic checkpoint complex(MCC). MCC and APC/C interact with each other so that the latter is in a closed conformation which means that it cannot be combined with substrate, leading to the fact that subsequent process cannot be carried out smoothly. The SAC signal is mainly produced from those chromosomes which have not been correctly captured by the spindle. Changes to the kinetochore components from the combination of chromosomes and spindle, abnormal microtubule assembly, the kinetochore microtubules combination being affected are all regarded as the sources of sustained activation of SAC signaling in cells. This paper mainly discussed the activation of SAC signal from the connection of granulocyte microtubule and CPC is an important compound in this process.CPC(Chromosomal passenger complex) is involved in many cellular activities during the period of mitosis and during the early stage, it is mainly located in the chromosome and jointly involved in mitotic early regulation with a series of periodic protein, after that, it has been transferred to the spindle center and is involved in the cytokinesis regulation. CPC is composed of its original core kinase Aurora B and the other three proteins(INCENP, Survivin and Borealin) which are responsible for regulation and localization.The INCENP is a connector of CPC complex assembly and its Amino-terminal is necessary for complex positioning and divided into a group of three bundles together with the related group of Survivin and Borealin which jointly determine the positioning of CPC in centromere(mitotic early stage) and in spindle center area(mitosis anaphases). While Aurora B is the core of CPC compound functioning, to be specific, it conducts the depolymerization of wrongly connected kinetochore and microtubules through the phosphorylation of corresponding substrate to reduce the tension of spindle. In the period of mitosis, cell requires an accurate chromosome segregation process which needs that kinetochore and spindle microtubules conduct a two-way and correct combination. The CPC complex plays an important role in the mismatch and repairing of kinetochore microtubule binding through its kinase unit Aurora B. At the same time, many unconnected kinetochores will be produced and this signal is recognized by SAC, which contributes to the activation of SAC signaling pathway.This paper discusses the phenomenon and mechanism of arrest during cell mitosis period caused by ROS activating SAC, finding out that ROS treated cells and the cells cannot move forward to the anaphase of mitosis due to the arrest in mitotic prophase and metaphase. To explore the mechanism, we found out that it has no effect on microtubule assembly but it indeed hindered the Aurora B kinase functioning in CPC complex so that we speculate that ROS affected the activity of Aurora B kinase and interfered with its function in kinetochore microtubules connection, as a result, the activation of SAC has been realized. This paper has proposed the function of ROS in activating SAC for the first time and the subsequent impact on mitosis process. What’s more, during the process of exploring its mechanism, the function of Aurora B kinase during the mitosis period of cells has also been investigated from another perspective. Research Objective: This paper aims to reveal the role of ROS in the process of cell mitosis, explore the effects of ROS on cells except the DNA oxidative damage and to find out its mechanism. Most of the previous reports and literatures focused on the oxidative damage to cells of ROS and the relationship with disease occurrence while we mainly study the effects of ROS on cell cycle progression, moreover, except its DNA oxidative damage in the interval, we also describe the effect of ROS on cell mitosis process during the mitosis period. Research Contents: this paper studies the effect of ROS on cell mitosis process, analyses the related phenomena and explores its mechanism. To be specific, we mainly research the effect of H2O2 on cell mitosis state and related components, and we also conduct the analysis of the function of CPC compound related to the activation of SAC. After that, we eventually concentrate on the activity of Aurora B kinase and further explore the mechanism of ROS. Experiment Methods: In our experiment, we use H2O2 and VK3 as the source of ROS to stimulate cells, staining the nucleus and using fluorescence microscopy to observe the state of cell nucleus, at the same time, conducting statistics of cell mitosis ratio in order to study the effect of ROS on cell mitosis process phenomenon. We use MTT kit to detect the tolerance situation of different cells against H2O2. During the process of exploring the mechanism, we use immune fluorescence method to observe the microtubule assembly and positioning of Aurora B while we use the immune blotting method to analyze the molecular level and modification level of relevant proteins. Experiment Results: Under the treatment of H2O2, some cells like Hela S3 came across mitotic arrest(different cells have different sensitivities to H2O2). VK3 can also have the similar effect on cells like H2O2. The effect of H2O2 on Aurora B kinase is significant, to be specific, the positioning has weakened in the centromere during the period of mitosis and the phosphorylation level has also decreased. At the same time, we found out that the phosphorylation level of INCENP protein which jointly constitutes CPC complexes with Aurora B has also decreased under the influence of H2O2. Experiment Conclusion: This paper studies the effect of ROS on cell mitosis process and a preliminary conclusion has been reached that ROS has affected the function of Aurora B kinase in the process of kinetochore microtubule binding and it has activated the mitotic spindle assembly checkpoint(SAC), finally leading to the mitotic arrest.