| Summary: | 碩士 === 國立清華大學 === 生命科學系 === 87 === Geldanamycin (GA), a benzoquinone ansamycin, is a potent inducer of ER stress pathway by affecting the folding of proteins in the ER. Herein, we show that exposure of 9L rat brain tumor cell to 5 mM GA leads to enhance the synthesis of the 78 kDa glucose-regulated protein (GRP78). The induction of GRP78 by GA is concentration- and time-dependent and this process coincides to the accumulation of its mRNA. We demonstrate for the first time that the intracellular level of reactive oxygen species (ROS) is significant increase in GA-treated cells and this process is inhibited by N-acetylcysteine (NAC) and partially by pyrrolidine dithiocarbamate (PDTC). Similar results can be found in the GRP78 protein and mRNA level. These data lead us to conclude that generation of ROS plays a major role in the GA-induced grp78 expression in 9L rat brain tumor cells. By using the electrophoretic mobility shift assay (EMSA), we showed that all of the three previous identified regulatory elements including CORE, CRE, and C1 are involved in the basal expression of GRP78. However, we found that the nuclear extracts prepared from GA-treated cells exhibit an significant increase in binding activity toward the cis-regulatory elements including CORE and C1. Moreover, this increase in binding activity toward the CORE element is reduced by NAC. We conclude that the oxidative stress is activated by GA treatment and this process is crucial in the GA-induced expression of GRP78 and the CORE element plays the important role in 9L RBT cells.
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