Stable transfection of PC12 cells with estrogen receptor (ERalpha): protective effects of estrogen on cell survival after serum deprivation.

Potential protective effects of the gonadal steroid estrogen on neurons are of particular interest in aging, neurodegenerative disease, and other traumatic conditions. In this study, we examined the hypothesis that estrogen, acting through the estrogen receptor (ERalpha), can enhance neuronal cell survival in the face of serious apoptotic challenge. PC12 cells were transfected with full-length rat ERalpha cDNA and a number of stable transfectants that expressed ER mRNA and protein (PCER cells) at levels comparable to those present in uterus or the MCF7 breast cancer cell line were obtained. A control line of cells transfected with vector DNA alone (PCCON cells) was used for comparisons. The apoptotic challenge used in the experiments was serum-free media, as it is well established that undifferentiated PC12 cells rapidly undergo cell death via apoptosis under those conditions. Estrogen treatment of PCER cells markedly increased the viability of these cells relative to PCCON cells in serum-free media, as assessed by trypan blue staining and TUNEL staining. We also examined the mitotic effects of estrogen treatment. While estrogen significantly stimulated bromodeoxy uridine (BrdU) incorporation into PCER cells in low-serum, but otherwise steroid-free media, no BrdU incorporation occurred in serum-free media. Mitotic effects of estrogen in low-serum steroid-free media were completely abolished by treatment with the estrogen receptor antagonist ICI 182,780. From this we conclude that the effects of estrogen on PCER cells in serum-free media can be attributed to increased cell survival, rather than proliferation.