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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ store depletion, and overexpression of ribosomal proteins define a novel phenotype of ER stress-associated multidrug resistance.