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

Zivadinovic D, Gametchu B, Watson CS - Breast Cancer Res. (2004)

Bottom Line: 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.ICI172,780 blocked the E2-induced response and 17alpha-estradiol did not elicit the response, also suggesting activity through an estrogen receptor.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA. ddzivadi@utmb.edu

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.

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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.
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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.

Mentions: We observed that the level and kinetics of cAMP accumulation varied depending on the type of incubation medium, the incubation temperature, and pretreatment with the PDE inhibitor IBMX. In mERhigh MCF-7 cells, 1 pmol/l E2 induced rapid and transient production of cAMP (Fig. 2a,2b,2c). At the reduced temperature of 4°C in a completely defined medium (Fig. 2a), a substantial increase in accumulated cAMP was seen as compared with the levels achieved at 37°C (Fig. 2b). The response peak at 4°C was prolonged, as the accumulated cAMP decreased gradually, even in the absence of the PDE inhibitor IBMX, which is usually included to inhibit the decay of cAMP and enhance the response. The same level of accumulated cAMP was obtained at 5, 15 and 30 min at 4°C, regardless of whether the treatment was performed with cells attached to a plate (Fig. 2a, open circles) or in suspension (Fig 2a, closed circles). However, after 30 min the cAMP level declined abruptly in attached cells. The DCSS medium did not increase cAMP production at 4°C (data not shown).


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

Zivadinovic D, Gametchu B, Watson CS - Breast Cancer Res. (2004)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064104&req=5

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.
Mentions: We observed that the level and kinetics of cAMP accumulation varied depending on the type of incubation medium, the incubation temperature, and pretreatment with the PDE inhibitor IBMX. In mERhigh MCF-7 cells, 1 pmol/l E2 induced rapid and transient production of cAMP (Fig. 2a,2b,2c). At the reduced temperature of 4°C in a completely defined medium (Fig. 2a), a substantial increase in accumulated cAMP was seen as compared with the levels achieved at 37°C (Fig. 2b). The response peak at 4°C was prolonged, as the accumulated cAMP decreased gradually, even in the absence of the PDE inhibitor IBMX, which is usually included to inhibit the decay of cAMP and enhance the response. The same level of accumulated cAMP was obtained at 5, 15 and 30 min at 4°C, regardless of whether the treatment was performed with cells attached to a plate (Fig. 2a, open circles) or in suspension (Fig 2a, closed circles). However, after 30 min the cAMP level declined abruptly in attached cells. The DCSS medium did not increase cAMP production at 4°C (data not shown).

Bottom Line: 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.ICI172,780 blocked the E2-induced response and 17alpha-estradiol did not elicit the response, also suggesting activity through an estrogen receptor.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA. ddzivadi@utmb.edu

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.

Show MeSH
Related in: MedlinePlus