INVOLVEMENT OF CALCIUM MEDIATED REACTIVE OXYGEN SPECIES IN INDUCTIVE GRP78 EXPRESSION BY GELDANAMYCIN IN 9L RAT BRAIN TUMOR CELLS

• Main Authors: Hsin-Yi Shyu, 徐心儀
• Other Authors: Prof. Yiu-Kay Lai
• Format: Others
• Language: en_US
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/08375949457903533397

碩士 === 國立清華大學 === 生命科學系 === 90 === Geldanamycin (GA), a benzoquinone ansamycin, is an inhibitor of HSP90 and has been implicated as a potent anti-cancer drug. Previous studies reported that endoplasmic reticulum stress (ER stress) was evoked with GA, and in our laboratory reactive oxygen species (ROS) was found to play important signal transduction roles. Glucose regulated protein 78 (GRP78) is mainly located in lumen of ER and has chaperone function to assist new synthesized proteins folding to correct conformations, and it was induced in response to ER stress. In this study, we investigated the roles of calcium, ROS and their interrelationship in GA-induced GRP78 expression in 9L rat brain tumor (RBT) cells. The induction of GRP78 by GA requires intact process of protein synthesis and is regulated mainly in transcription level. By monitoring cytoplasmic calcium contents with fluorescence microscopy, the calcium signaling evoked by GA was influenced by depletion of organelle-stored calcium or by preventing the influx of calcium across the plasma membrane. Chelation of intracellular calcium with 1,2-bis(2-amino- phenoxy)ethane-N,N,N’,N’,-tetraacetic acid (BAPTA/AM) or extracellular calcium with ethylene glyco-bis(β-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid (EGTA) reduces GRP78 induction in GA-treated 9L RBT cells, implying that calcium signaling is required for GRP78 induction. The compound 1-96-[17beta- 3-methoxyestra-1,3,5(10)-trien-17-yl]-aminohexyl)-1H-pyrrole-2,5-dione (U73122) decreases the GA-induced GRP78 expression, suggesting that this calcium mobilization might be dependent on phospholipase C. To assess the possible kinases involved, inhibitors were used and coupled with calcium monitoring. We found that the increase of cytoplasmic calcium concentration in GA signal transduction pathway may act through protein kinase C (PKC) and the specific subtype involved may be PKCβII. Significant reduction of GRP78 induction was also evident when genistein was applied, indicating the involvement of protein tyrosine kinase. On the other hand, antioxidants prevent GRP78 induction by GA, implying ROS generation in vivo under GA treatment. To distinguish the contribution of diverse chemical species of ROS, our results indicate that GA-induced GRP78 expression was prominently blocked by antioxidants specific for hydroxyl radical. To delineate the causal effects of these intermediates, we show that increase in cytoplasmic calcium concentration activating PKCbII precedes the generation of ROS. In conclusion, we investigated the regulation of inductive expression of GRP78 in 9L RBT cells by GA treatment and identified that this signal transduction pathway is acting through calcium, PLC, PKCβII, and protein tyrosine kinase and then driving the ROS generation to concert this induction.