Stable transfection of PC12 cells with estrogen receptor (ERα): Protective effects of estrogen on cell survival after serum deprivation

Abstract

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.

Melatonin and oestrogen treatments were able to improve neuroinflammation and apoptotic processes in dentate gyrus of old ovariectomized female rats

“…Replacement with 17β-oestradiol (21 days) throughout the post-ovariectomy period reduced the number of apoptotic cells to control levels and prevented the enhancing effects of ovariectomy on Bax expression but only partially restored Bcl-2 expression. It has been shown that oestradiol reduced mRNA levels of the pro-apoptotic Bad in PC12 cells transfected with ERα (Gollapudi and Oblinger 1999). Nopparat et al (2010) have demonstrated, in the SK-N-SH dopaminergic cell line, a novel role of melatonin in protecting cells from autophagic cell death triggered by the Bcl-2/Beclin 1 pathway, by inhibiting the activation of the JNK1 (cJun amino-terminal kinase), Bcl-2 upstream pathway.…”

Estrogen receptor-α and -β immunoreactivity and mRNA in neurons of sensory and autonomic ganglia and spinal cord

“…Such effects could involve protein synthetic activity and help explain the volume changes reported by Coujard (1951). In addition, Gollapudi and Oblinger (1999a) have shown that ER-α is present in PC12 cells [neural crest derivatives that are nerve growth factor (NGF)-dependent] and that estrogens have cytoprotective (cell survival) effects that involves NGF and the antiapoptotic molecule Bcl-X L (Gollipudi and Oblinger 1999b). Furthermore, estrogens stimulate neurotransmitter levels (in CNS neurons; Gibbs et al 1994;Okamura et al 1994aOkamura et al , 1994bYuri and Kawata 1994;Dufourny and Warembourg 1999;Miller et al 1999), upregulate neuronal-nitric oxide synthase in autonomic and/or sensory nerves in the vagina (Berman et al 1998), or downregulate CGRP in sensory neurons (Moussaoui et al 1996;Duval et al 1998;Yang et al 1998).…”

Diethyl Phthalate Enhances Apoptosis Induced by Serum Deprivation in PC12 Cells

“…DNA fragmentation occurs via the action of endonucleases. The TUNEL method specifically labels DNA ends generated by endonuclease activity [28]. To confirm the results of DNA electrophoresis, quantification of DNA ladders was carried out by the TUNEL methods in the cells treated with 0-10,000 ng/ ml DEP for 72 hr.…”