Diethylstilbestrol-Induced Estrogen Receptor-Dependent [Ca2+]i Rises and Apoptosis in Chinese Hamster Ovary (CHO) Cells

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca2+ concentrations ([Ca2+]i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations ≥ 1∝ increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. In Ca2+-free medium, after pretreatment with 50∝ DES, 1∝ thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor)-induced [Ca2+]i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca2+]i rises. At a concentration of 5∝, DES increased cell viability. At concentrations of 100–200 μ M, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 μM DES on viability was partly reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′ -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 μ M)-induced [Ca2+]i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 μ M). ICI-182,780 did not affect 5 μ M DES-induced increase in viability but partly reversed 100 μ M DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca2+]i rises by stimulating estrogen receptors leading to Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca2+-dependent pathway.