The study of modulating effects on X-ray induced cell mutability by chaperons and SUMO.

我们之前曾报道，人血清因子对细胞突变性的调节与蛋白水解酶活性水平的提高有关。本工作旨在研究细胞突变性与GRP78、GRP94、HSP27和SUMO-3等伴侣之间的关系。：(1)GRP94，其中一个伴侣基因GRP94通过X射线诱导的蛋白水解酶分析，确定为该介体分子的候选基因。通过对GRP94 mRNA的siRNA进行克隆形成实验，推测GRP94参与了细胞对X射线的抗性。(2)通过紫外线诱导的蛋白酶分析，我们还鉴定了其他伴侣HSP27。当HSP27的表达受到抑制时，紫外光照射后6-4个光产物的去除活性降低。我们进一步证实GRP78也参与了紫外线损伤的修复机制。因此，伴侣对细胞突变性的调控可能是通过蛋白水解酶信号途径实现的。(3)泛素样蛋白SUMO-3被鉴定为X射线照射后DNA合成活性异常的候选基因。已知SUMO-3的功能受特定的蛋白水解酶的调节。当SUMO-3的表达降低时，X射线照射后DNA合成活性增加。我们表达了GFP-SUMO-3融合蛋白，并观察到X射线照射后细胞核内有明显浓度的绿色荧光。应用一种新的方法来评估人血清中的氧化应激。通过测定血清过氧化氢来监测氧化应激状态。部分患者和重度吸烟者的血清过氧化氢水平高于健康志愿者。

We previously reported that modulation of cell mutability by human serum factors is associated with increased levels of protease activity. The present work is designed to examine the relationships between cell mutability and chaperons such as GRP78,GRP94,HSP27 and SUMO-3. : (1)GRP94 ; One of the chaperons, GRP94, was identified as a candidate gene for the mediator molecule by analysis of X-ray-induced protease. GRP94 was suggested to be involved in cellular X-ray resistance by the colony forming assay using siRNA for GRP94 mRNA.(2)We also identified other chaperons, HSP27, by analysis of UV-induced protease. The activity of removing 6-4 photo products after UV irradiation was decreased when expression of HSP27 was suppressed. We further identified that GRP78 is also involved in the repair mechanism for the UV-induced damage. Thus, the modulation of cell mutability by chaperons seems to be mediated though protease signaling.(3)One of ubiquitin-like proteins, SUMO-3, was identified as a candidate gene for abnormal DNA synthesis activity after X-ray irradiation. It is known that the function of SUMO-3 is regulated by specific proteases. The activity of DNA synthesis after X-ray irradiation was increased when expression of SUMO-3 was decreased. We expressed GFP-SUMO-3 fusion protein and observed a marked concentration of green fluorescence in the nuclear after X-ray irradiation.Application of a new method to evaluate oxidative stress in human serum. A test to monitor the oxidative stress by evaluating serum hydroperoxide was carried out. Serum hydrpperoxide of some patients and heavy-smokers showed higher levels than healthy volunteers.