Membrane estrogen receptor-alpha levels in MCF-7 breast cancer cells predict cAMP and proliferation responses - PubMed (original) (raw)
Comparative Study
doi: 10.1186/bcr958. Epub 2004 Nov 24.
Affiliations
- PMID: 15642158
- PMCID: PMC1064104
- DOI: 10.1186/bcr958
Comparative Study
Membrane estrogen receptor-alpha levels in MCF-7 breast cancer cells predict cAMP and proliferation responses
Dragoslava Zivadinovic et al. Breast Cancer Res. 2005.
Abstract
Introduction: 17beta-estradiol (E2) 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 (mERlow) and high (mERhigh) expression of the membrane form of estrogen receptor (mER)-alpha, 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 mERhigh (mER-alpha-enriched) and mERlow (mER-alpha-depleted) populations. Unique (compared with previously reported) incubation conditions at 4 degrees C were found to be optimal for demonstrating E2-induced cAMP production. Time-dependent and dose-dependent effects of E2 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 E2-induced cell proliferation were assessed using the crystal violet assay.
Results: We demonstrated a rapid and transient cAMP increase after 1 pmol/l E2 stimulation in mERhigh cells; at 4 degrees C these responses were much more reliable and robust than at 37 degrees C (the condition most often used). The loss of cAMP at 37 degrees 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 mERhigh cells than in mERlow cells, implicating mER-alpha levels in the process. ICI172,780 blocked the E2-induced response and 17alpha-estradiol did not elicit the response, also suggesting activity through an estrogen receptor. E2 dose-dependent cAMP production, although biphasic in both cell types, was responsive to 50-fold higher E2 concentrations in mERhigh cells. Proliferation of mERlow cells was stimulated over the whole range of E2concentrations, whereas the number of mERhigh 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-alpha participates. E2-mediated activation of adenylyl cyclase and downstream participation of protein kinase A were shown to be involved in these responses.
Conclusion: Rapid mER-alpha-mediated nongenomic signaling cascades generate cAMP and downstream signaling events, which contribute to the regulation of breast cancer cell number.
Figures
Figure 1
Verification of the utility of the crystal violet (CV) assay for measuring cell number. (a) Different numbers of MCF-7 cells enriched for membrane estrogen receptor-α (mERhigh) and MCF-7 cells depleted for membrane estrogen receptor-α (mERlow; 1000–7000/well) were plated, fixed with 2% paraformaldehyde/0.1% glutaraldehyde, and then quantified via the CV assay at 590 nm absorbance. (b) Different numbers of mERhigh cells were assessed in parallel with CV and MTT assays to demonstrate a linear correlation between these assays. The values are means for 28 samples ± standard error.
Figure 2
Optimization of conditions for 17β-estradiol (E2)-induced cAMP accumulation and measurement. (a–c) MCF-7 cells enriched for membrane estrogen receptor-α (mERhigh) and (d, e) MCF-7 cells depleted for membrane estrogen receptor-α (mERlow). All of the cells were stimulated with 1 pmol/l E2, or an equivalent amount of E2 conjugated to peroxidase, for different time intervals, and the intracellular cAMP levels were assessed. (Panels a and d) Cells were incubated at 4°C in defined medium (DM) either attached to a plate (open circles) or in suspension (closed circles). (Panels b and e) Attached cells were incubated at 37°C in DM medium (triangles) or DCSS medium (medium with 4 × dextran-coated charcoal-stripped serum; squares). (Panel c) Cells in suspension were stimulated with E2-peroxidase at 4°C. All experiments were repeated at least three times, and each time point was in triplicate. The data are presented as means ± standard error and the asterisks represent significant differences (P < 0.05) as compared with time 0.
Figure 3
Kinetics of cAMP decrease in MCF-7 cells enriched for membrane estrogen receptor-α (mERhigh) cells. (a) Cells were treated with 1 pmol/l 17β-estradiol (E2) for different time intervals at 37°C in the presence of 1 mmol/l 3-isobutyl-1-methylxanthine (IBMX). The intracellular cAMP (circles) and that in the medium (squares) from the same cells were assessed. (b) cAMP was produced by directly stimulating adenylyl cyclase with 10 μmol/l forskolin for 15 min at 37°C. The decrease in cAMP in the cytosol was tested at 37°C in the absence (open circles) and presence of 1 mmol/l IBMX (closed circles). The entire regression lines were compared by evaluating the differences between the sums of squares of the residuals of individual lines with the sum of squares of the residuals of the combined line using an F-test. The data are presented as means ± standard error. The regression lines were significantly different (P = 0.0001).
Figure 4
Effects of ICI172,780 and 17α-estradiol on cAMP production in MCF-7 cells enriched for membrane estrogen receptor-α (mERhigh) cells. (a) ICI172,780 inhibited 17β-estradiol (E2)-induced cAMP production. Both simultaneous application of 1 μmol/l ICI172,780 and 1 pmol/l E2 (open circle) or 30 min pretreatment with ICI172,780 (open square) was tested. (b) 17α-Estradiol was applied at a 10 nmol/l concentration and the time dependent cAMP production was followed. Closed circles represent E2 induced cAMP production, redrawn for comparison from Fig. 2a.
Figure 5
17β-Estradiol (E2) dose-dependent cAMP responses and proliferation in MCF-7 cells enriched for membrane estrogen receptor-α (mERhigh; closed circles) versus MCF-7 cells depleted for membrane estrogen receptor-α (mERlow; open circles). (a) cAMP response; each experiment was repeated three times with triplicate samples, and the data were approximated with a four-parameters Gaussian equation. The two sets of data were compared by evaluating the differences between the sums of squares of the residuals from each individual curve with the sum of squares of the residuals of the combined curve using the F-test. The curves were significantly different (P = 0.02). (b) E2 dose-dependent proliferation curves vary with mER expression level. mERhigh cells (closed circles) exhibited a biphasic growth pattern, whereas mERlow cells (open circles) responded with a sigmoid growth pattern. The cells were treated on separate plates with different concentrations of E2 or ethanol vehicle (0.1%) for 5 days. The data are percentages of control, and expressed as mean values ± standard error from three separate experiments, each containing 40 replicates.
Figure 6
Effect of cell density on 17β-estradiol (E2)-induced proliferation. Different densities of MCF-7 cells enriched for membrane estrogen receptor-α (mERhigh; shaded bars) and MCF-7 cells depleted for membrane estrogen receptor-α (mERlow; open bars) were treated for 5 days with 10 nmol/l E2. The controls were treated on a separate plate with ethanol vehicle (0.1%). Values are expressed as percentage change from control. All experimental mean values (error bars are ± standard error) were significantly different (P < 0.002) from the control (first bar on the left).
Figure 7
Signaling pathway inhibitors differentially affect cell proliferation. 17β-Estradiol (E2; 10 nmol/l) was introduced on day 0. Adenylyl cyclase stimulator (forskolin; 10 μmol/l) and cAMP analog (8-CPT cAMP; 250 μmol/l) were replenished every second day. PKA inhibitor (H-89; 10 μmol/l) and adenylyl cyclase inhibitor (SQ 22,536; 300 μmol/l) were applied at day 0 together with 10 nmol/l E2. Cells were fixed on days 1, 3 and 5, and their number estimated by crystal violet (CV) assay. The data are averaged values ± standard error (error ranges were very small and are contained within the size of the symbol) of two separate experiments, each containing 24 replicates. mERhigh, MCF-7 cells enriched for membrane estrogen receptor-α; mERlow, MCF-7 cells depleted for membrane estrogen receptor-α.
Figure 8
Proliferation of cells after 5 days of treatment with different compounds. The same incubation conditions apply as for Fig. 7. The asterisks indicate significant differences (P < 0.002) from controls. Data are expresssed as means ± standard error. E2, 17β-estradiol; mERhigh, MCF-7 cells enriched for membrane estrogen receptor-α; mERlow, MCF-7 cells depleted for membrane estrogen receptor-α.
Figure 9
Possible negative feedback effects of estrogen-activated cAMP-PKA (protein kinase A) pathway on the estrogen-activated Ras-Raf-MAPK (mitogen-activated protein kinase) pathway of cell proliferation. The up arrow indicates a cAMP increase. AC, adenylyl cyclase; E2, 17β-estradiol; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase.
References
- Stumpel F, Scholtka B, Jungermann K. Stimulation by portal insulin of intestinal glucose absorption via hepatoenteral nerves and prostaglandin E2 in the isolated, jointly perfused small intestine and liver of the rat. Ann N Y Acad Sci. 2000;915:111–116. - PubMed
- Lohmann SM, Fischmeister R, Walter U. Signal transduction by cGMP in heart. Basic Res Cardiol. 1991;86:503–514. - PubMed
- Albrecht ED, Pepe GJ. Placental steroid hormone biosynthesis in primate pregnancy. Endocr Rev. 1990;11:124–150. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Miscellaneous