Mutagenesis of HEK293 Cells Reveals Phenotypic and Genotypic Variants Involved in Hsp90‐Mediated Glucocorticoid Signaling

Abstract

Hsp90 is a highly conserved eukaryotic chaperone protein responsible for mediating a multitude of intracellular signaling pathways, including those associated with the glucocorticoid receptor (GR), hsp70, and stress response. The goal of this study was to investigate the effects of mutagenesis on human embryonic kidney (HEK293) cells in the presence of a GR agonist (dexamethasone) as well as an hsp90 antagonist and clinically relevant benzoquinone ansamycin (17‐AAG, a geldanamycin derivative). HEK293 cells were incubated with increasing concentrations of dexamethasone and 17‐AAG in order to determine cell growth inhibition and cytotoxicity. Cell lysate was prepared when growth approached 80% confluency and analyzed for hsp90 and hsp70 via western blot. Relative concentrations for the proteins were qualitatively measured via ImageJ analysis software. The concentration of each drug necessary for producing an effect in hsp90 concentration while not exhibiting a significant decrease in cell viability was determined. HEK293 cell growth was assayed over 7 days in the presence of one or both drugs and then cultured in increasing concentrations of 17‐AAG in order to select for a drug resistant phenotype. The growth characteristics, phenotype, and genotype of drug resistant cells were then examined. Western blot analysis determined a significant increase in hsp90 concentration at 10 μM dexamethasone and an increase in hsp70 concentration in cell populations at all drug conditions tested relative to control. The growth curve performed at 10 μM dexamethasone reflected a disruption in normal cell cycle progression. A concentration of 1 μM 17‐AAG was shown to significantly decrease the concentration of hsp90 and 10 μM of the drug was determined to be cytotoxic. Growth characteristics of cells exposed to increasing concentrations of 17‐AAG over an extended time period were quantified, and exons of HSP90AA1 and HSP90AB1 were assayed via Sanger sequencing. Increases in hsp90 concentrations in the presence of dexamethasone and possible genetic alterations following prolonged 17‐AAG incubations presumptively indicate cells with directional hsp90 regulation can better survive exposure to high concentrations of clinically relevant glucocorticoids as well as hsp90 inhibitors.