Membrane estrogen receptor-α levels in MCF-7 breast cancer cells predict cAMP and proliferation responses

INTRODUCTION: 17β-estradiol (E(2)) can rapidly induce cAMP production, but the conditions under which these cAMP levels are best measured and the signaling pathways responsible for the consequent proliferative effects on breast cancer cells are not fully understood. To help resolve these issues, we compared cAMP mechanistic responses in MCF-7 cell lines selected for low (mER(low)) and high (mER(high)) expression of the membrane form of estrogen receptor (mER)-α, and thus addressed the receptor subform involved in cAMP signaling. METHODS: MCF-7 cells were immunopanned and subsequently separated by fluorescence activated cell sorting into mER(high )(mER-α-enriched) and mER(low )(mER-α-depleted) populations. Unique (compared with previously reported) incubation conditions at 4°C were found to be optimal for demonstrating E(2)-induced cAMP production. Time-dependent and dose-dependent effects of E(2 )on cAMP production were determined for both cell subpopulations. The effects of forskolin, 8-CPT cAMP, protein kinase A inhibitor (H-89), and adenylyl cyclase inhibitor (SQ 22,536) on E(2)-induced cell proliferation were assessed using the crystal violet assay. RESULTS: We demonstrated a rapid and transient cAMP increase after 1 pmol/l E(2 )stimulation in mER(high )cells; at 4°C these responses were much more reliable and robust than at 37°C (the condition most often used). The loss of cAMP at 37°C was not due to export. 3-Isobutyl-1-methylxanthine (IBMX; 1 mmol/l) only partially preserved cAMP, suggesting that multiple phosphodiesterases modulate its level. The accumulated cAMP was consistently much higher in mER(high )cells than in mER(low )cells, implicating mER-α levels in the process. ICI172,780 blocked the E(2)-induced response and 17α-estradiol did not elicit the response, also suggesting activity through an estrogen receptor. E(2 )dose-dependent cAMP production, although biphasic in both cell types, was responsive to 50-fold higher E(2 )concentrations in mER(high )cells. Proliferation of mER(low )cells was stimulated over the whole range of E(2)concentrations, whereas the number of mER(high )cells was greatly decreased at concentrations above 1 nmol/l, suggesting that estrogen over-stimulation can lead to cell death, as has previously been reported, and that mER-α participates. E(2)-mediated activation of adenylyl cyclase and downstream participation of protein kinase A were shown to be involved in these responses. CONCLUSION: Rapid mER-α-mediated nongenomic signaling cascades generate cAMP and downstream signaling events, which contribute to the regulation of breast cancer cell number.