Abstract
A large number of correlative studies have established that the activation of the unfolded protein response (UPR) alters the cell's sensitivity to chemotherapeutic agents. Although the induction of the glucose-regulated proteins (GRPs) is commonly used as an indicator for the UPR, the direct role of the GRPs in conferring resistance to DNA damaging agents has not been proven. We report here that without the use of endoplasmic reticulum (ER) stress inducers, specific overexpression of GRP78 results in reduced apoptosis and higher colony survival when challenged with topoisomerase II inhibitors, etoposide and doxorubicin, and topoisomerase I inhibitor, camptothecin. While investigating the mechanism for the GRP78 protective effect against etoposide-induced cell death, we discovered that in contrast to the UPR, GRP78 overexpression does not result in G1 arrest or depletion of topoisomerase II. Caspase-7, an executor caspase that is associated with the ER, is activated by etoposide. We show here that specific expression of GRP78 blocks caspase-7 activation by etoposide both in vivo and in vitro, and this effect can be reversed by addition of dATP in a cell-free system. Recently, it was reported that ectopically expressed GRP78 and caspases-7 and -12 form a complex, thus coupling ER stress to the cell death program. However, the mechanism of how GRP78, a presumably ER lumen protein, can regulate cytosolic effectors of apoptosis is not known. Here we provide evidence that a subpopulation of GRP78 can exist as an ER transmembrane protein, as well as co-localize with caspase-7, as confirmed by fluorescence microscopy. Co-immunoprecipitation studies further reveal endogenous GRP78 constitutively associates with procaspase-7 but not with procaspase-3. Lastly, a GRP78 mutant deleted of its ATP binding domain fails to bind procaspase-7 and loses its protective effect against etoposide-induced apoptosis.
Highlights
A large number of correlative studies have established that the activation of the unfolded protein response (UPR) alters the cell’s sensitivity to chemotherapeutic agents
Quantitation of the immunoblots of whole cell extracts showed 5-fold higher GRP78 level in C.1 cells compared with the parental Chinese hamster ovary (CHO) cells, whereas the level of GRP94, an endoplasmic reticulum (ER)-localized chaperone protein, and a 45-kDa unidentified protein (X) recognizable by the anti-KDEL antibody was relatively constant in both cell lines (Fig. 1A)
To examine directly whether specific overexpression of GRP78 can lead to the development of drug resistance, CHO and C.1 cells were exposed to various drugs, and cell survival was measured using clonogenic survival assays
Summary
Cell Culture Conditions—The establishment of C.1 and AD-1 cell lines that are derivatives of CHO overexpressing wild-type or mutated hamster GRP78 has been described [9]. Annexin V Staining and FACS Analysis—CHO, C.1, and T24/83 cells were trypsinized, washed twice with ice-cold PBS, pH 7.4, and resuspended in 1ϫ binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of 1 ϫ 106 cells/ml. The immunoprecipitate was released from the washed beads by the addition of 30 l of 1ϫ SDS-PAGE sample loading buffer (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol), followed by heating at 100 °C for 10 min. The pellet, which represents ER membrane, was rinsed with cold water and resuspended in 1ϫ SDS-PAGE sample loading buffer and analyzed by Western blot. The pellet was washed three times with acetone, air-dried, solubilized in the 1ϫ SDS-PAGE sample loading buffer, and analyzed by Western blot. The proteolytic cleavage reactions were terminated by the addition of 1ϫ SDS-PAGE sample loading buffer and boiling at 100 °C for 5 min. 10-20 g of total protein from each reaction was analyzed by Western blot
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