Increased Calcium Influx and Ribosomal Content Correlate with Resistance to Endoplasmic Reticulum Stress-induced Cell Death in Mutant Leukemia Cell Lines

Abstract

Cell clones were derived by treatment of HL-60 cells with stepwise increasing concentrations of econazole (Ec), an imidazole antifungal that blocks Ca 2؉ influx and induces endoplasmic reticulum (ER) stress-related cell death in multiple mammalian cell types. Clones exhibit 20-to more than 300-fold greater resistance to Ec. Unexpectedly, they also display stable cross-resistance to tunicamycin, thapsigargin, dithiothreitol, and cycloheximide but not doxorubicin, etoposide, or Fas ligand. Phenotypic analysis indicates that the cells display increased store-operated calcium influx and resistance to ER Ca 2؉ store depletion by Ec. E2R2, the most resistant clone, was observed to maintain protein synthesis levels after treatment with Ec or thapsigargin. Expression of GRP78, an ER-based chaperone, was induced by these ER stress treatments but to equal degrees in HL-60 and E2R2 cells. By using microarray analysis, at least 15 ribosomal protein genes were found to be overexpressed in E2R2 compared with HL-60 cells. We also found that ribosomal protein content was increased by 30% in E2R2 as well as other clones. The resistance phenotype was partially reversed by the ribosome-inactivating protein saporin. Therefore, increased store-operated calcium influx, resistance to ER Ca 2؉ store depletion, and overexpression of ribosomal proteins define a novel phenotype of ER stress-associated multidrug resistance.Calcium signals play a central role in many cellular activities including cell movement, secretion, proliferation, gene transcription, and cell death (1, 2). In non-excitable cells, the endoplasmic reticulum (ER) 1 plays a key role in calcium signaling. Receptor-mediated activation of phospholipase C generates the second messenger inositol 1,4,5-triphosphate that diffuses rapidly through the cytosol to interact with inositol 1,4,5-triphosphate receptor on the ER membrane releasing calcium stored in the ER lumen. The resulting depletion of calcium within the ER triggers calcium entry from the extracellular milieu through the plasma membrane. This capacitive or store-operated calcium (SOC) influx (3) serves to both expand the initial calcium signal and replenish the emptied ER store (4, 5). Although the nature of SOC channel remains unclear, the importance of SOC influx in maintaining cell viability has been demonstrated in different studies (6 -13).In addition to its key role in Ca 2ϩ signaling, the ER is a site for protein synthesis, and these two functions intersect under conditions of ER stress. For instance, treatment of cells with thapsigargin (Tg), the inhibitor of the sarcoplasmic/endoplasmic Ca 2ϩ -ATPase responsible for transporting Ca 2ϩ into the ER, rapidly depletes ER Ca 2ϩ stores (14), inhibits the function of Ca 2ϩ -dependent chaperones, and triggers the unfolded protein response (15). Econazole (Ec) is an antifungal imidazole that depletes Ca 2ϩ from the ER of mammalian cells and blocks Ca 2ϩ influx (16, 17). These effects result in sustained depletion of Ca 2ϩ from ER stores and profound inhibition of prot...

Trichosanthin induced apoptosis in HL-60 cells via mitochondrial and endoplasmic reticulum stress signaling pathways

“…3B-D), which strongly supported the notion that ER stress signaling pathway is involved in the TCS-induced apoptosis in HL-60 cells. Interestingly, one recent study demonstrated that in ER stress-resistant HL-60 cells, many ribosomal proteins were upregulated and saporin, another RIP, can partially reverse the ER stress-resistant phenotype [31], implicating that RIP may impact ER function and cause or facilitate ER stress which is consistent with our finding. However, it is unclear how TCS may induce ER stress.…”

Inhibition of Ca2+Influx Is Required for Mitochondrial Reactive Oxygen Species-Induced Endoplasmic Reticulum Ca2+Depletion and Cell Death in Leukemia Cells

“…1, A to D, increased fluorescence levels, indicating increases in OS levels, was observed for cells treated with Ec but not Tg or Tu. We have previously demonstrated that all three agents induce ER stress within the chosen 2-h time period, as indicated by induction of eukaryotic translation initiation factor 2 subunit ␣ phosphorylation and BiP expression, along with suppression of protein synthesis Zhang and Berger, 2004). However, this is achieved differ- ently for each agent.…”

“…Preconditioning with sublethal levels of ER stress has been shown to protect cells, in part through up-regulation of chaperones (Liu et al, 1998;Hung et al, 2003). However, sustained ER stress will eventually result in prolonged protein synthesis inhibition that leads to cell death Zhang and Berger, 2004). Important mediators of ER stress-associated death include the cleavage and activation of the ER-associated caspase-12 (Szegezdi et al, 2003) and increased expression of CHOP/GADD153 (Wang et al, 1996), a transcription factor that sensitizes cells to apoptosis.…”

“…However unlike Tg, Ec additionally blocks Ca 2ϩ influx, resulting in sustained ER Ca 2ϩ depletion (Franzius et al, 1994;Jan et al, 1999;. In previous studies, we have shown that the Ca 2ϩ depletion caused by Ec induces activation enhanced cell death in leukemic cells, breast cancer cells, and murine bone marrow-derived mast cells through sustained inhibition of protein synthesis (Gommerman and Berger, 1998;Zhang et al, 2002;Zhang and Berger, 2004). However, the mechanism of how this compound affects the intracellular ER store and the subsequent fate of the cell remains unknown.…”

Release of intracellular calcium primes chronic myeloid leukaemia cells for tyrosine kinase inhibitor-induced apoptosis

“…[7][8][9][10] Rises in both cytosolic and mitochondrial calcium have been reported in several cell types to lead to apoptosis. [11][12][13][14][15] BCR-ABL expression has been reported to modulate calcium homeostasis in the ER, and interfere with ER-mediated apoptotic pathways to promote survival of CML cells. 16 Changes in either the prevailing ER calcium levels and/or resultant calcium influx into the ER (known as capacitative calcium entry (CCE)) may compromise calcium-dependent apoptotic mechanisms in BCR-ABL-expressing cells.…”