Yeast CUP1 protects HeLa cells against copper-induced stress

As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. Yeast CUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study, CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P作为一种必需的微量元素，铜在过量蓄积时会对哺乳动物细胞产生毒性。金属硫蛋白（Metallothioneins，MTs）是一类富含半胱氨酸的小型蛋白质，可强效结合铜离子，因此在重金属解毒过程中发挥关键作用。酵母CUP1属于MT基因家族成员。本研究旨在探究酵母CUP1能否有效结合铜离子并保护细胞抵御铜胁迫。本研究通过实时荧光定量PCR（quantitative real-time PCR）检测CUP1的表达水平，采用电感耦合等离子体质谱法（inductively coupled plasma mass spectrometry）测定细胞内铜含量；利用2',7'-二氯二氢荧光素二乙酸酯（2',7'-dichlorofluorescein-diacetate, DCFH-DA）法检测细胞内活性氧（reactive oxygen species, ROS）的生成量；采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide，简称MTT）法检测细胞活力；通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分析CUP1过表达细胞的细胞周期分布。实验数据显示，在HeLa细胞中过表达酵母CUP1可对铜离子诱导的细胞胁迫产生保护作用，提升细胞存活率（P