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Membrane estrogen receptor-alpha levels predict estrogen-induced ERK1/2 activation in MCF-7 cells.

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

Bottom Line: The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-alpha (membrane plus intracellular receptors).These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol (E2), as well as different patterns of E2 dose-dependent responsiveness.Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A.

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: We examined the participation of a membrane form of estrogen receptor (mER)-alpha in the activation of mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK]1 and ERK2) related to cell growth responses in MCF-7 cells.

Methods: We immunopanned and subsequently separated MCF-7 cells (using fluorescence-activated cell sorting) into mER-alpha-enriched (mERhigh) and mER-alpha-depleted (mERlow) populations. We then measured the expression levels of mER-alpha on the surface of these separated cell populations by immunocytochemical analysis and by a quantitative 96-well plate immunoassay that distinguished between mER-alpha and intracellular ER-alpha. Western analysis was used to determine colocalized estrogen receptor (ER)-alpha and caveolins in membrane subfractions. The levels of activated ERK1 and ERK2 were determined using a fixed cell-based enzyme-linked immunosorbent assay developed in our laboratory.

Results: Immunocytochemical studies revealed punctate ER-alpha antibody staining of the surface of nonpermeabilized mERhigh cells, whereas the majority of mERlow cells exhibited little or no staining. Western analysis demonstrated that mERhigh cells expressed caveolin-1 and caveolin-2, and that ER-alpha was contained in the same gradient-separated membrane fractions. The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-alpha (membrane plus intracellular receptors). These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol (E2), as well as different patterns of E2 dose-dependent responsiveness. The maximal kinase activation was achieved after 10 min versus 6 min in mERhigh versus mERlow cells, respectively. After a decline in the level of phosphorylated ERKs, a reactivation was seen at 60 min in mERhigh cells but not in mERlow cells. Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A.

Conclusion: Our results suggest that the levels of mER-alpha play a role in the temporal coordination of phosphorylation/dephosphorylation events for the ERKs in breast cancer cells, and that these signaling differences can be correlated to previously demonstrated differences in E2-induced cell proliferation outcomes in these cell types.

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Optimization of 96-well plate immunoassay for membrane estrogen receptor (mER) quantification in MCF-7 cells. (a) Different concentrations of estrogen receptor-α-specific C-542 antibody (1–12 μg/ml) were tested. Enzymatic paranitrophenol phosphate (pNpp) hydrolysis yielding the paranitrophenol (pNp) product was monitored at 37°C for 5, 15 and 30 min, as shown. Cells pretreated for 72 hours with medium containing dextran-coated charcoal-stripped serum (DCSS) versus defined medium (DM) are represented with closed and open circles, respectively. (b) Antibody binding compared in nonpermeabilized (open bars) and permeabilized (shaded bars) conditions. no1°no2° represents primary and secondary antibodies omitted; no1° represents primary antibody omitted (only secondary antibody applied); mIgG1k represents mouse IgG1k isotype control; peptide comp. represents C-542 epitope peptide blocking of the interaction with primary antibody; anti-clathrin represents our test for cell membrane integrity. Values are expressed as means ± standard error. CV, crystal violet.
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Figure 4: Optimization of 96-well plate immunoassay for membrane estrogen receptor (mER) quantification in MCF-7 cells. (a) Different concentrations of estrogen receptor-α-specific C-542 antibody (1–12 μg/ml) were tested. Enzymatic paranitrophenol phosphate (pNpp) hydrolysis yielding the paranitrophenol (pNp) product was monitored at 37°C for 5, 15 and 30 min, as shown. Cells pretreated for 72 hours with medium containing dextran-coated charcoal-stripped serum (DCSS) versus defined medium (DM) are represented with closed and open circles, respectively. (b) Antibody binding compared in nonpermeabilized (open bars) and permeabilized (shaded bars) conditions. no1°no2° represents primary and secondary antibodies omitted; no1° represents primary antibody omitted (only secondary antibody applied); mIgG1k represents mouse IgG1k isotype control; peptide comp. represents C-542 epitope peptide blocking of the interaction with primary antibody; anti-clathrin represents our test for cell membrane integrity. Values are expressed as means ± standard error. CV, crystal violet.

Mentions: To quantify mER-α with our plate assay, we first determined the lowest optimal C-542 antibody concentration that would saturate binding to the membrane form of the receptor, and then optimized assay conditions to give a reliably detectable signal in the linear range of the fluorescent product assay. All three signal generation incubation times (5, 15 and 30 min) gave acceptable data, but 15 min assays allowed clear delineation of antigen-saturating concentrations of C-542 antibody utilizing lower antibody concentrations (8 μg/ml; Fig. 4a). Those conditions were used for subsequent assays. The same level of mER-α could be detected in cells kept for 3 days in a completely defined medium lacking serum or in the same medium supplemented with 10% DCSS (Fig. 4a, open circles and closed circles, respectively). The background values (obtained in the absence of primary and secondary antibody, or with no primary antibody) were very low (Fig. 4b). At 8 μg/ml the isotype control mIgG1k yielded a value only 10% of that for C-542 antibody; peptide competition resulted in an approximate 50% decrease in C-542 antibody binding, again verifying the specificity of the antibody for this epitope. This verified that the assay was specific, and not subject to interference by endogenous alkaline phosphatases in the presence of levamasole. The fixation conditions that we used preserved the integrity of the cell membrane, as demonstrated by the negligible anti-clathrin antibody binding in nonpermeabilized versus the very high binding in permeabilized cells. Therefore, equivalent protocols differing only in the inclusion of a permeabilization agent during fixation can be used to quantify the total ER (tER) versus the mER.


Membrane estrogen receptor-alpha levels predict estrogen-induced ERK1/2 activation in MCF-7 cells.

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

Optimization of 96-well plate immunoassay for membrane estrogen receptor (mER) quantification in MCF-7 cells. (a) Different concentrations of estrogen receptor-α-specific C-542 antibody (1–12 μg/ml) were tested. Enzymatic paranitrophenol phosphate (pNpp) hydrolysis yielding the paranitrophenol (pNp) product was monitored at 37°C for 5, 15 and 30 min, as shown. Cells pretreated for 72 hours with medium containing dextran-coated charcoal-stripped serum (DCSS) versus defined medium (DM) are represented with closed and open circles, respectively. (b) Antibody binding compared in nonpermeabilized (open bars) and permeabilized (shaded bars) conditions. no1°no2° represents primary and secondary antibodies omitted; no1° represents primary antibody omitted (only secondary antibody applied); mIgG1k represents mouse IgG1k isotype control; peptide comp. represents C-542 epitope peptide blocking of the interaction with primary antibody; anti-clathrin represents our test for cell membrane integrity. Values are expressed as means ± standard error. CV, crystal violet.
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Related In: Results  -  Collection

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Figure 4: Optimization of 96-well plate immunoassay for membrane estrogen receptor (mER) quantification in MCF-7 cells. (a) Different concentrations of estrogen receptor-α-specific C-542 antibody (1–12 μg/ml) were tested. Enzymatic paranitrophenol phosphate (pNpp) hydrolysis yielding the paranitrophenol (pNp) product was monitored at 37°C for 5, 15 and 30 min, as shown. Cells pretreated for 72 hours with medium containing dextran-coated charcoal-stripped serum (DCSS) versus defined medium (DM) are represented with closed and open circles, respectively. (b) Antibody binding compared in nonpermeabilized (open bars) and permeabilized (shaded bars) conditions. no1°no2° represents primary and secondary antibodies omitted; no1° represents primary antibody omitted (only secondary antibody applied); mIgG1k represents mouse IgG1k isotype control; peptide comp. represents C-542 epitope peptide blocking of the interaction with primary antibody; anti-clathrin represents our test for cell membrane integrity. Values are expressed as means ± standard error. CV, crystal violet.
Mentions: To quantify mER-α with our plate assay, we first determined the lowest optimal C-542 antibody concentration that would saturate binding to the membrane form of the receptor, and then optimized assay conditions to give a reliably detectable signal in the linear range of the fluorescent product assay. All three signal generation incubation times (5, 15 and 30 min) gave acceptable data, but 15 min assays allowed clear delineation of antigen-saturating concentrations of C-542 antibody utilizing lower antibody concentrations (8 μg/ml; Fig. 4a). Those conditions were used for subsequent assays. The same level of mER-α could be detected in cells kept for 3 days in a completely defined medium lacking serum or in the same medium supplemented with 10% DCSS (Fig. 4a, open circles and closed circles, respectively). The background values (obtained in the absence of primary and secondary antibody, or with no primary antibody) were very low (Fig. 4b). At 8 μg/ml the isotype control mIgG1k yielded a value only 10% of that for C-542 antibody; peptide competition resulted in an approximate 50% decrease in C-542 antibody binding, again verifying the specificity of the antibody for this epitope. This verified that the assay was specific, and not subject to interference by endogenous alkaline phosphatases in the presence of levamasole. The fixation conditions that we used preserved the integrity of the cell membrane, as demonstrated by the negligible anti-clathrin antibody binding in nonpermeabilized versus the very high binding in permeabilized cells. Therefore, equivalent protocols differing only in the inclusion of a permeabilization agent during fixation can be used to quantify the total ER (tER) versus the mER.

Bottom Line: The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-alpha (membrane plus intracellular receptors).These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol (E2), as well as different patterns of E2 dose-dependent responsiveness.Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A.

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: We examined the participation of a membrane form of estrogen receptor (mER)-alpha in the activation of mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK]1 and ERK2) related to cell growth responses in MCF-7 cells.

Methods: We immunopanned and subsequently separated MCF-7 cells (using fluorescence-activated cell sorting) into mER-alpha-enriched (mERhigh) and mER-alpha-depleted (mERlow) populations. We then measured the expression levels of mER-alpha on the surface of these separated cell populations by immunocytochemical analysis and by a quantitative 96-well plate immunoassay that distinguished between mER-alpha and intracellular ER-alpha. Western analysis was used to determine colocalized estrogen receptor (ER)-alpha and caveolins in membrane subfractions. The levels of activated ERK1 and ERK2 were determined using a fixed cell-based enzyme-linked immunosorbent assay developed in our laboratory.

Results: Immunocytochemical studies revealed punctate ER-alpha antibody staining of the surface of nonpermeabilized mERhigh cells, whereas the majority of mERlow cells exhibited little or no staining. Western analysis demonstrated that mERhigh cells expressed caveolin-1 and caveolin-2, and that ER-alpha was contained in the same gradient-separated membrane fractions. The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-alpha (membrane plus intracellular receptors). These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol (E2), as well as different patterns of E2 dose-dependent responsiveness. The maximal kinase activation was achieved after 10 min versus 6 min in mERhigh versus mERlow cells, respectively. After a decline in the level of phosphorylated ERKs, a reactivation was seen at 60 min in mERhigh cells but not in mERlow cells. Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A.

Conclusion: Our results suggest that the levels of mER-alpha play a role in the temporal coordination of phosphorylation/dephosphorylation events for the ERKs in breast cancer cells, and that these signaling differences can be correlated to previously demonstrated differences in E2-induced cell proliferation outcomes in these cell types.

Show MeSH
Related in: MedlinePlus